Conditions: Childhood Mortality
Keywords: Childhood mortality Azithromycin Mass treatment Vaccine Visit targeted treatment
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 77664
Healthy volunteers: yes

Brief summary:
An estimated 7.7 million pre-school aged children die each year, the majority from infectious diseases. Mass azithromycin distributions for trachoma may have the unintended benefit of reducing childhood mortality. We recently demonstrated the biannual mass azithromycin distribution significantly reduces all-cause child mortality in a cluster randomized trial (MORDOR I) conducted in three diverse regions of Sub-Saharan Africa.

Our long-term goal is to more precisely define the role of mass azithromycin treatments as an intervention for reducing childhood morbidity and mortality. We propose a cluster randomized trial designed to repeat the original study to confirm the original results in a different geographic study with similarly high child mortality, and to better understand the mechanism behind any effect of azithromycin on child mortality. We hypothesize that biannual mass azithromycin distribution will reduce child mortality compared to placebo, and that this effect will be primarily driven by a reduction in infectious burden.

Objectives:

1. Determine the efficacy of biannual mass azithromycin distribution versus placebo in children aged 1-59 months for reduction in all-cause mortality.
2. Determine the efficacy of targeted azithromycin distribution to infants during an early infant healthcare visit (approximately 5th through 12th week of life) on infant mortality.
3. Determine the mechanism behind the effect of biannual mass azithromycin distribution for reduction in child mortality.

The study will be conducted in the Nouna District in northwestern Burkina Faso.

Detailed description:
Although child health and mortality are improving worldwide, children in the Sahel and sub-Sahel regions of West Africa have the greatest risks of mortality.Burkina Faso's current under-5 mortality rate is estimated 110 per 1,000 live births. Similar to other countries in the region, the major causes of child mortality in Burkina Faso are malaria, respiratory tract infection, and diarrhea. Malnutrition acts as a major underlying contributor to mortality. Interventions that address these underlying causes may be particularly efficacious for reducing mortality.

Younger children at are at a higher risk of mortality. Approximately 2/3rd of under-5 deaths occur during the first year of life. In general, the child mortality rate decreases as age increases. While some improvement has been observed, neonatal mortality is declining at a slower rate than post-neonatal childhood mortality. Many child health interventions are designed specifically for children over 6 months of age, such as vitamin A supplementation, seasonal malaria chemoprevention, and lipid-based nutritional supplementation. Identification of strategies that are safe and effective for the youngest children will be required to address persistently high rates of neonatal and infant mortality.

The MORDOR I study demonstrated a significant reduction in all-cause child mortality following biannual mass azithromycin distribution. Across three diverse geographic locations in sub-Saharan Africa (Malawi, Niger, and Tanzania), biannual mass azithromycin distribution over a two-year period led to a 14% decrease in all-cause child mortality. In Niger, 1 in 5-6 deaths were averted. These results are qualitatively similar to those of a previous study of mass azithromycin distribution for trachoma control in Ethiopia, which found reduced odds of all-cause mortality in children in communities receiving mass azithromycin compared to control communities.

In MORDOR I, the strongest effect of azithromycin was in the youngest cohort of children. Across all three countries, the strongest effect of azithromycin was consistently in children 1-5 months of age, with an approximately 25% reduction in all-cause mortality. However, MORDOR I was not optimized to target the youngest age groups. Although children as young as 1 month were eligible, biannual distributions might not reach some children until 7 months of age. On average, children were first treated at 4 months. Given that there may be a substantial benefit to treating children at younger ages, azithromycin strategies that are designed to target younger age groups may be even more beneficial for reducing child mortality.

Here, we propose a randomized controlled trial designed to evaluate the efficacy of mass and targeted azithromycin strategies for child mortality. In the rural northwestern district of Nouna in Burkina Faso, we propose to randomize villages to biannual mass azithromycin distribution or placebo. This study was designed by CRSN and UCSF partners to confirm the results of MORDOR I, evaluate an alternative health systems distribution point (the vaccine visit) for delivery of azithromycin to young children, and to provide a platform for evaluation of potential mechanisms behind the effect of azithromycin by collecting and processing additional specimens and tests.

Objectives:

1. Determine the efficacy of biannual mass azithromycin distribution versus placebo in children aged 1-59 months for reduction in all-cause mortality.
2. Determine the efficacy of targeted azithromycin distribution to infants during an early infant healthcare visit (approximately 5th through 12th week of life) on infant mortality.
3. Determine the mechanism behind the effect of biannual mass azithromycin distribution for reduction in child mortality.

Study Design:

CRSN and UCSF (hereafter, "we") will assess childhood mortality over three years, comparing communities where children aged 1-59 months receive biannual oral azithromycin and/or targeted azithromycin during the 5th-12th week of life in conjunction with the first Expanded Programme on Immunization (EPI) vaccine visit or biannual placebo and targeted placebo. All eligible communities in Nouna District will be randomized (278 communities). A random sample of 48 (12/arm) communities from within the HDSS will be selected to participate in the "Mortality Plus" study, which will entail an annual morbidity exam among 15 randomly selected children per community to monitor infectious disease morbidity, nutritional status, and macrolide resistance. All communities will contribute to the mortality outcome.

Arms:
arm 1: Bi-annual Mass Azithromycin distribution to all children 1-60 months old in participating communities arm 2: Bi-annual Mass Placebo distribution to all children 1-60 months old in participating communities arm 3: Targeted placebo to children 5 to 12 weeks old at vaccine visit or other healthy child visit arm 4: Targeted azithromycin to children 5 to 12 weeks old at vaccine visit or other healthy child visit

Interventions: intervention 1: Azithromycin intervention 2: Placebos
Intervention descriptions: intervention 1: biannual azithromycin in eligible communities to children 1 to 59 months old Targeted azithromycin to children aged 5 to 8 weeks old at the vaccine visit intervention 2: biannual placebo in eligible communities to children 1 to 59 months old Targeted placebo to children aged 5 to 8 weeks old at the vaccine visit

Protocol document:
Prot_002.pdf: 
version 2.5 
 
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Community Health Azithromycin Trial  
Burkina Faso  
 
Manual of Operations and Procedures 
 
Centre de Recherche en Santé de Nouna 
 
The 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 
Guillaume Compaoré, MD 
Alphonse Zakane, MS 
Mamadou Ouattara, MD 
Valentin Boudo 
Till Bärnighausen, MD ScD 
Thuy D Doan, MD, PhD 
Jeremy Keenan, MD MPH 
Benjamin Arnold, PhD 
Travis Porco, PhD MPH 
Kieran O’Brien, MPH 
Elodie Lebas, RN 
Jessica Brogdon, MPH 
Catherine Cook, MPH 
Ariana Austin, MS 
William Godwin, MPH 
Ying Lin, MSPH 
Fanice Nyatigo, BS 
Huiyu Hu, MS 
 
 
Last update: 04/18/2022 
 
 
 


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Table of Contents 
ABBREVIATIONS ................................................................................................................................. 6 
CHAPTER 1: OVERVIEW .............................................................................................................. 7 
1.1. 
EXECUTIVE SUMMARY ........................................................................................................................ 7 
1.2. 
OBJECTIVES ...................................................................................................................................... 7 
1.3. 
STUDY PARTNERS............................................................................................................................... 7 
1.4. 
STUDY SITE ....................................................................................................................................... 8 
CHAPTER 2: CONTEXT ............................................................................................................... 10 
CHAPTER 3: STUDY DESIGN..................................................................................................... 12 
3.1. 
RANDOMIZATION ............................................................................................................................. 12 
CHAPTER 4: STUDY ELIGIBILITY ............................................................................................. 14 
4.1. 
ELIGIBLE COMMUNITIES .................................................................................................................... 14 
ELIGIBLE INDIVIDUALS ..................................................................................................................................... 15 
4.2. 
STUDY SCHEDULE ............................................................................................................................. 16 
CHAPTER 5: CORE AND NON-CORE STUDY ELEMENTS .................................................... 17 
5.1. 
MORTALITY STUDY – ALL COMMUNITIES ............................................................................................. 17 
5.1.1. 
Core Elements ...................................................................................................................... 17 
5.2. 
MORTALITY PLUS COMMUNITIES ........................................................................................................ 19 
5.2.1. 
Core Elements ...................................................................................................................... 19 
5.2.2. 
Non-core Study Elements .................................................................................................. 20 
5.3 INDIVIDUALLY RANDOMIZED TARGETED TREATMENT ........................................................................................ 20 
CHAPTER 6:  CENSUS ................................................................................................................. 22 
6.1. 
CENSUS.......................................................................................................................................... 22 
6.2. 
RANDOM CENSUS VERIFICATION ........................................................................................................ 25 
6.3. 
VERBAL AUTOPSY ..................................................................................................................... 25 
6.4. 
VALIDATING THE MORTALITY OUTCOME ............................................................................... 26 
CHAPTER 7: REGISTERING PARTICIPANTS FOR SPECIMEN COLLECTION ................... 27 
CHAPTER 8: BLOOD SAMPLES ................................................................................................. 27 
8.1. 
FINGERSTICK ............................................................................................................................. 28 
8.2. 
DRIED BLOOD SPOTS FOR SEROLOGY .................................................................................... 29 
SET-UP DRYING AREA FOR TROPBIO BLOODSPOTS ................................................................................................. 31 
8.3. 
HEMOGLOBIN TEST .................................................................................................................. 31 
8.4. 
THICK AND THIN SMEARS FOR MALARIA .............................................................................. 32 
8.5. 
MATERIALS FOR BLOOD COLLECTION ................................................................................................... 34 
CHAPTER 9: SPECIMEN COLLECTION FOR RESISTANCE TESTING ................................. 35 
9.1. 
POPULATION ................................................................................................................................... 35 
9.2 NASOPHARYNGEAL SWABS ......................................................................................................................... 35 
9.2.1 
Materials for Swab Collection for Resistance Testing ................................................. 36 
9.2.2 
Protocol for Tubing and Handling of Samples ............................................................... 36 
9.3 
STOOL SAMPLES ................................................................................................................................... 37 
9.3.1 
Stool Specimen Collection ................................................................................................. 38 
9.3.2 
Materials for Stool Specimen Collection ........................................................................ 39 
9.3.3 Protocol for Fresh Frozen Stool Collection .......................................................................... 40 


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9.4 
QUALITY CONTROL MEASURES FOR SPECIMEN COLLECTION ......................................................................... 42 
CHAPTER 10: TRAINING ............................................................................................................ 42 
10.1 
STANDARDIZATION ........................................................................................................................... 42 
CHAPTER 11: SAMPLE ORGANIZATION, TRANSPORT, AND STORAGE ......................... 42 
11.1 
DE-IDENTIFICATION .......................................................................................................................... 43 
11.2 
SPECIMEN TRANSPORT ............................................................................................................. 43 
11.3 
SPECIMEN STORAGE ................................................................................................................. 44 
11.3.1 
Short-term Sample Storage........................................................................................... 44 
11.3.2 
Long-term Sample Storage ........................................................................................... 44 
11.4 
CATALOG SPECIMENS............................................................................................................... 44 
CHAPTER 12: STUDY MEDICATION ........................................................................................ 44 
12.1 STUDY MEDICATION DESCRIPTION (FROM PFIZER, INC.) ................................................................................ 45 
12.2 
DOSAGE INFORMATION ..................................................................................................................... 45 
12.3 
MEDICATION PROCUREMENT/DONATION ............................................................................................ 45 
12.4 
MEDICATION QUALITY CONTROL ........................................................................................................ 45 
12.5 
ANTIBIOTIC DISTRIBUTION & MONITORING COVERAGE........................................................................... 45 
12.6 
ADVERSE REACTIONS/SIDE EFFECTS .................................................................................................... 46 
12.7 
ADVERSE EVENTS SYSTEMS ................................................................................................................ 47 
12.7.1 
Passive Adverse Events Monitoring ........................................................................... 47 
12.7.2 
Active Adverse Events Monitoring.............................................................................. 47 
12.7.3 
Adverse Events Data ..................................................................................................... 49 
12.8  SUPPLY ISSUES....................................................................................................................................... 50 
CHAPTER 13: PROTECTION OF HUMAN SUBJECTS ............................................................. 51 
13.1 
INSTITUTIONAL REVIEW BOARD APPROVAL ........................................................................................... 51 
13.2 
INFORMED CONSENT ........................................................................................................................ 52 
13.3 
RISKS AND BENEFITS OF STUDY PROCEDURES ........................................................................................ 52 
13.3.1 
Verbal Autopsy ............................................................................................................... 52 
13.3.2 
Swabbing Procedures..................................................................................................... 52 
13.3.3 
Stool Collection .............................................................................................................. 52 
13.3.4 
Blood Testing .................................................................................................................. 53 
13.3.5 
Anthropometric Measurements.................................................................................... 53 
CHAPTER 14: STUDY MONITORING ....................................................................................... 54 
CHAPTER 15: DATA AND SAFETY MONITORING COMMITTEE CHARTER .................... 54 
15.1 
PRIMARY RESPONSIBILITIES OF THE DSMC ........................................................................................... 54 
15.2 
DSMC MEMBERSHIP................................................................................................................ 55 
15.3 
CONFLICTS OF INTEREST .......................................................................................................... 55 
15.4 
TIMING AND PURPOSE OF THE DSMC MEETINGS ................................................................ 55 
15.5 
PROCEDURES TO ENSURE CONFIDENTIALITY AND PROPER COMMUNICATION .............................................. 56 
15.6 
STATISTICAL MONITORING GUIDELINES ................................................................................................ 57 
15.7 
DSMC CONTACT INFORMATION......................................................................................................... 58 
CHAPTER 16: DATA COLLECTION, MANAGEMENT, AND SECURITY.............................. 59 
16.1 
SCOPE OF DATA ............................................................................................................................... 59 
16.2 
DATA STORAGE, MANAGEMENT, AND SECURITY......................................................................... 59 
16.3 
DATA MONITORING AND CLEANING .......................................................................................... 60 


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APPENDIX .................................................................................................................................... 61 
 


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1 
Abbreviations 
 
CRSN: Centre de Recherche en Santé de Nouna 
DCC: Data Coordinating Center  
GPS: global positioning system 
IRB: Institutional Review Board 
MUAC: mid-upper arm circumference  
NP swabs: nasopharyngeal swabs 
PCR: polymerase chain reaction  
STGG: skim milk tryptone glucose glycerin media 
UCSF: University of California San Francisco  
WHO: World Health Organization 
DHMT: District Health Management Team 


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2 Chapter 1: Overview 
 
1.1. 
Executive Summary 
An estimated 7.7 million pre-school aged children die each year, the majority 
from infectious diseases.1 Mass azithromycin distributions for trachoma may 
have the unintended benefit of reducing childhood mortality.1 We recently 
demonstrated the biannual mass azithromycin distribution significantly reduces 
all-cause child mortality in a cluster randomized trial (MORDOR I) conducted in 
three diverse regions of Sub-Saharan Africa.2  
 
Our long-term goal is to more precisely define the role of mass azithromycin 
treatments as an intervention for reducing childhood morbidity and mortality. 
We propose a cluster randomized trial designed to repeat the original study to 
confirm the original results in a different geographic study with similarly high 
child mortality, and to better understand the mechanism behind any effect of 
azithromycin on child mortality. 
 
1.2. 
Objectives  
 
1: Determine the efficacy of biannual mass azithromycin distribution 
versus placebo in children aged 1-59 months for reduction in all-cause 
mortality. We hypothesize that biannual distribution of azithromycin will lead to 
significantly reduced all-cause mortality among children aged 1-59 months after 
36 months of treatment. 
 
2: Determine the efficacy of targeted azithromycin distribution to infants 
during an early infant healthcare visit (approximately 5th through 12th 
week of life) on infant mortality. We hypothesize that infants receiving a 
single dose of azithromycin during early post-neonatal infancy will have 
significantly lower all-cause mortality compared to infants receiving placebo. 
 
3: Determine the mechanism behind the effect of biannual mass 
azithromycin distribution for reduction in child mortality.  
 
1.3. 
Study Partners 
This study was jointly designed and will be jointly implemented by partners at 
CRSN and UCSF. CRSN and UCSF partners contributed equally to the development 
of this protocol. Funding for the study is provided by the Bill and Melinda Gates 
Foundation. 


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1.4. 
Study Site 
The study will be conducted in the Nouna District in northwestern Burkina Faso. 
It is situated about 300 kilometres north-west of Ouagadougou, the capital of 
Burkina Faso. 
Nouna Health District is one of the six districts of Boucle du Mouhoun Health 
Region and covers the geographical area of Kossi Province in the western part of 
the country. Nouna is the capital of Kossi province. The health district comprises 
of the town of Nouna with a total population of 29,297 inhabitants and a rural 
area of about 235,426 inhabitants. 
The multicultural society consists of 15 ethnic groups whereof the major ones are 
the Mossi,  Bwaba, Marka, Samo, Gourounsi and Peuhl ethnic groups. The main 
socioeconomic activity of the population in the district is farming, similar to the 
rest of the country. The only exceptions are the Peuhl who are semi-nomadic 
cattle herders and dairy producers. 
The health infrastructure consists of one District Hospital in Nouna (Centre 
Medical avec Antenne chirurgicale, CMA), and over 34 dispensaries (Centre de 
Santé et de Promotion Sociale, CSPS). The District Hospital in Nouna covers a 
population of 300,360 inhabitants. The Health Centers each cover a total 
population between 2,195 and 34,581 inhabitants. Due to deficient road 
infrastructure only 69.13% of health facilities are accessible for the DHMT all 
over the year with a mean distance of 8.48 kms (Nouna District action plan 2012). 
Targeted treatment will also be done in the district of K. Vigué in the Haut 
Bassins region and in the district of Banfora in the Cascades region.. 
 
 


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3  


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4 Chapter 2: Context 
 
Although child health and mortality are improving worldwide, children in the 
Sahel and sub-Sahel regions of West Africa have the greatest risks of mortality.4,5 
Burkina Faso’s current under-5 mortality rate is estimated 110 per 1,000 live 
births4. Similar to other countries in the region, the major causes of child 
mortality in Burkina Faso are malaria, respiratory tract infection, and diarrhea. 
Malnutrition acts as a major underlying contributor to mortality.6,7 Interventions 
that address these underlying causes may be particularly efficacious for reducing 
mortality. 
 
Younger children are at a higher risk of mortality. Approximately 2/3rd of 
under-5 deaths occur during the first year of life.4 In general, the child mortality 
rate decreases as age increases. While some improvement has been observed, 
neonatal mortality is declining at a slower rate than post-neonatal childhood 
mortality.4 Many child health interventions are designed specifically for children 
over 6 months of age, such as vitamin A supplementation, seasonal malaria 
chemoprevention, and lipid-based nutritional supplementation. Identification of 
strategies that are safe and effective for the youngest children will be required to 
address persistently high rates of neonatal and infant mortality. 
 
The MORDOR I study demonstrated a significant reduction in all-cause child 
mortality following biannual mass azithromycin distribution. Across three 
diverse geographic locations in sub-Saharan Africa (Malawi, Niger, and 
Tanzania), biannual mass azithromycin distribution over a two-year period led 
to a 14% decrease in all-cause child mortality. In Niger, 1 in 5-6 deaths were 
averted. These results are qualitatively similar to those of a previous study of 
mass azithromycin distribution for trachoma control in Ethiopia, which found 
reduced odds of all-cause mortality in children in communities receiving mass 
azithromycin compared to control communities.1  
 
In MORDOR I, the strongest effect of azithromycin was in the youngest cohort 
of children. Across all three countries, the strongest effect of azithromycin was 
consistently in children 1-5 months of age, with an approximately 25% reduction 
in all-cause mortality. However, MORDOR I was not optimized to target the 
youngest age groups. Although children as young as 1 month were eligible, 
biannual distributions might not reach some children until 7 months of age. On 
average, children were first treated at 4 months. Given that there may be a 
substantial benefit to treating children at younger ages, azithromycin strategies 


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that are designed to target younger age groups may be even more beneficial for 
reducing child mortality.    
 
Here, we propose a randomized controlled trial designed to evaluate the 
efficacy of mass and targeted azithromycin strategies for child mortality. In the 
rural northwestern district of Nouna in Burkina Faso, we propose to randomize 
villages to biannual mass azithromycin distribution or placebo. This study was 
designed by CRSN and UCSF partners to confirm the results of MORDOR I, 
evaluate an alternative health systems distribution point (targeted treatment) for 
delivery of azithromycin to young children, and to provide a platform for 
evaluation of potential mechanisms behind the effect of azithromycin by 
collecting and processing additional specimens and tests. 


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5 Chapter 3: Study Design 
 
The research team will assess childhood mortality over three years, comparing 
communities where children aged 1-59 months receive biannual oral 
azithromycin and/or targeted azithromycin during the 5th-12th week of life in 
conjunction with the first Expanded Programme on Immunization (EPI) vaccine 
visit, the BCG vaccine visit and the 42-day postnatal health visit/well-child visit 
or biannual placebo and targeted placebo. All eligible communities in Nouna 
District will be randomized (278 communities). A random sample of 48 (24/arm) 
communities from within the HDSS will be selected to participate in the 
“Mortality Plus” study, which will entail an annual morbidity exam among 15 
randomly selected children per community to monitor infectious disease 
morbidity, nutritional status, and macrolide resistance. All communities will 
contribute to the mortality outcome. All biologic specimens collected as part of 
the morbidity and resistance outcomes will be stored and made available to other 
investigators for further laboratory testing at the conclusion of the study, per 
Gates Foundation guidelines. 
 
 
3.1. 
Randomization  
 
Randomization of Treatment Allocation. All eligible communities in Nouna 
District will be randomized in a 1:1 fashion to biannual azithromycin or placebo. 
Targeted treatment will be randomized 1:1 individually to azithromycin or 
placebo in the nouna, K vigué and banfora districts. Refer to SAP for 
randomization details.  
 
Study Participants: At months 0, 12, 24, and 36 a random sample of children will 
be selected using a computer-generated simple random sample for exams and 
sample collection to monitor for morbidity and resistance in the Mortality Plus 
communities.


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Figure 1: Trial Profile 
 
 
 
 


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6 
Chapter 4: Study Eligibility   
 
4.1. 
Eligible Communities 
To be eligible for the trial, a community must meet the following criteria: 
 
1. The community location in target district. 
2. The community leader consents to participation in the trial (this does 
not obviate the need for individual consent, but without overall 
leadership consent, the community as a whole cannot be part of the 
trial).  
3. Eligible communities estimated population no more than 2,000 people. 
All communities with an estimated population of more than 2000 
people will be split into 2 or more randomization units 
4. The community is not in an urban area.  
 


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6.1 
Eligible Individuals 
 
Mortality Study:  
Census: The study can be thought of as consisting of seven 6-month 
segments, each of which starts with a census and ends with a follow-up 
census. All children in the study communities aged 1-59 months (up to but 
not including the 5th birthday) at the initial census of each segment are 
eligible to participate in the subsequent 6-month segment of the study. 
Note that the information for children erroneously entered into the census 
(e.g., children younger than 1 month or ≥ 60 months) can be corrected at 
the subsequent treatment, subsequent census, or at a verbal autopsy later 
in the study. However, these changes will not be applied retroactively; 
these misclassified children will still be included in the study population 
for that 6-month segment and any deaths will be counted toward the 
primary outcome. In addition, all children listed on the initial census for 
that 6-month segment will be included in the outcome, regardless of 
whether they received the study drug. 
 
Treatment: Individuals allergic to macrolides or azalides will not be given 
the study antibiotic azithromycin, but will be included in the outcome. 
Children weighing less than 3.8 kg will not be given treatment either.  
 
Birth Notification: All births in all study communities will be recorded 
over the duration of the study.   
 
Mortality Plus Communities:  
 
Census: The criteria for being included in the census are the same as the 
Mortality Study, as described above. 
 
Treatment: The inclusion and exclusion criteria for treatment are the same 
as for the Mortality Study, as described above. 
 
Examination & Sample Collection: A random sample of children aged 1-
59 months (up to but not including the 5th birthday) are eligible for 
examination and sample collection. As described above, the random 
sample will be a simple random sample based on the previous census. 
These individuals will likewise be selected from the previous census. 
 
 
 
 


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4.2. 
Study Schedule 
The schedule for examination and treatment is shown below in Table 1:  
MORDOR II Study Schedule 
 
 
 
  
MORTALITY 
MORTALITY PLUS 
Ongoing 
Birth notification 
1st EPI or BCG or 42-day postnatal 
visit/well-child visit azithro or placebo 
6 mo vital status 
6 mo Anthropometry assessments 
Birth notification 
1st EPI or BCG or 42-day post natal/well-
child visit azithro or placebo 
6 mo vital status 
6 mo Anthropometry assessments 
MORDOR 0 
Aug-19 to Jan-19 
Census 
Azithro or placebo 
Census 
Swabs  
Blood 
Anthropometry  
Azithro or placebo 
MORDOR 6 
Feb-19 to Jul-20 
 
Census 
Verbal Autopsy 
Azithro or placebo 
Census 
Azithro or placebo 
MORDOR 12 
Aug-20 to Jan-20 
Census 
Verbal Autopsy 
Azithro or placebo 
Census 
Azithro or placebo 
MORDOR 18 
Feb-20 to Jul-21 
Census 
Verbal Autopsy 
Azithro or placebo 
Census 
Azithro or placebo  
MORDOR 24 
Aug-21 to Jan-21 
Census 
Verbal Autopsy 
Azithro or placebo  
Census 
Azithro or placebo  
MORDOR 30 
Feb-21 to Jul-22 
Census 
Verbal Autopsy 
Azithro or placebo  
Census 
Azithro or placebo  
MORDOR 36 
Feb-22 to Jul-22 
Census 
Verbal Autopsy 
Azithromycin for all  
Birth History (subset of communities 
 
Census 
Swabs 
Blood 
RDT 
Anthropometry 
Swab targeted treatment group 
Note: Only children aged 1 month to 59 months in each community will be treated during mass drug administration 
 


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7 Chapter 5: Core and Non-Core Study Elements 
An overview of core and non-core elements for the mortality and morbidity study is 
provided here, but will be described in more detail in the following chapters.  
5.1. 
Mortality Study – All Communities 
 
5.1.1. Core Elements  
We will conduct the following study activities for the mortality study: 
Training 
Standardization activities before each biannual census will consist of didactic 
classroom instruction and mock census activities, followed by in-field training.  
Pre-census and mapping questionnaire 
Before the beginning of the study, we will perform a pre-census to be able to list all 
compounds and household in our study area. This pre-census will help us organize 
our data census collection by knowing the area we will be working on. 
The pre-census will include all questions ask during regular census (see chapter 
census below) and will include a mapping questionnaire about the characteristics of 
each household: 
- 
Wall, roof and ground of the house 
- 
Type of water supply 
- 
Latrinization 
- 
Electricity  
- 
Telephone (if exist take number with permission) 
- 
Cooking  
- 
Education of the head of household and mother/guardian 
- 
Child individual information: 
o Pre schooling  
o Breastfeeding  
o Use of bednet 
o Handicap 
 
Census 
An enumerated population census for 0 – 60 month olds (focusing on this age group) 
will be conducted every 6 months by trained field workers masked to study arm, 
recording births, deaths, and migration of children eligible for treatment. Pregnant 


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women will be noted at each census, to maximize inclusion of newborns on the 
subsequent census. 
Core Census Elements 
The following elements will be considered part of the core HDSS census 
activities:  
• Enumeration of the compound 
• Full enumeration of household members with emphasis on children aged 
0-60 months (age, sex) in a way that each child could be link to his mother 
• Enumeration of pregnancies  
• Recording of caregiver for each child <60 months 
• Recording head of household 
• Mid-upper arm circumference measurement for all children aged 0-60 
months 
• GPS coordinates 
 
Random Census Verification 
A repeat census will be conducted in a random selection of households at each study 
visit. Personnel will be different from the original census. This will allow us to assess 
whether the census identified all births, deaths, and migratory episodes relative to 
the previous census. 
VerbalAutopsy 
Verbal autopsy will be conducted in the district according to current methods for 
measuring verbal autopsy for all children aged 1-59 months who died during the 
study.3 The interview is conducted with the caregivers or relatives, using the 
WHO standard verbal autopsy questionnaire WHO-VA-2016. The interview 
usually takes place two months after the event with the person who assisted the 
deceased before the death. The data collected will be coded using InterVA 4. 
 
Treatment 
Children aged 1-59 months on the current census will be offered weight- or height-
based (<1 year and older children who can’t stand will be weighted; ≥1 year will be 
measured with a flexible dosing stick), directly observed, oral azithromycin 
suspension (or oral placebo) every 6 months for 3 years as performed in trachoma 
programs. Specifically, individuals are eligible on or after their one month birthday, 
and prior to the day of their fifth birthday.  
At the final treatment distribution, CHAT 36, all children 1-59 months old will be 
given a single dose of azithromycin. Placebo will not be utilized at the final phase.  


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Antibiotic Coverage Surveillance 
We will estimate antibiotic coverage from the most recent biannual census records. 
At the end of each treatment round at months 6, 12, 18, 24, and 30 we will identify 
any children who have missed 2 or more consecutive treatments, and forward this 
information to the census team. 
Birth history at the 36 months visit 
At the final visit (36 month) we will obtain birth history in a subset of communities. 
All women in childbearing age living in selected communities will be asked if they 
experienced births in the last 10 years. Name of women, age, name of children and 
age or date of birth will be collected. Vital status of each birth will be recorded. 
5.2. 
Mortality Plus Communities 
 
5.2.1. Core Elements 
 
All core elements described in 5.1.1 will also be conducted in the Mortality Plus 
communities.   
 
The designation “core elements” means that all study communities will be 
performing the study activity.  
 
In all study sites, we will perform the following tests on a random sample of 15 
children aged 1-60 months from each community at baseline, 12, 24, and 36 
months:  
- 
Blood samples (dried blood spots) for malaria and anemia 
- 
Nasopharyngeal swabs for pneumococcal macrolide resistance 
- 
Stool samples or rectal swabs to assess for macrolide resistance 
- 
Anthropometric assessments 
- 
Rapid diagnostic test for malaria (at 36 months only) 
 
Samples will be processed at the CRSN laboratory for microbiological culture, 
targeted PCR, serologic, thin and thick smears, and ova and parasite tests. 
Samples for microbiome analyses and specialized tests required by the Gates 
Foundation will be shipped to the United States. 
 
At the final visit (36 months), in the mortality plus communities, we will obtain 
one rectal swab from up to 10 children per community who participated in the 
targeted treatment distribution. 
 
 


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5.2.2. Non-core Study Elements 
The designation “non-core” element means that not all study communities will 
be participating in the activity.  
 
Mother MUAC assessment 
In a subset of 20 villages, we will train mothers/guardians on measuring MUAC 
on their children weekly. We will train mothers/guardians to bring children to 
the nearest CSPS for evaluation if MUAC is <125mm. 
 
Passive Surveillance 
In each Centre de Santé et de Promotion Sociale (CSPS, community health facility), 
we will conduct morbidity passive surveillance.  Each CSPS will be equipped with a 
tablet for electronic capture of health facility visits. Each visit will be recorded, 
including the reason for the visit (e.g., fever, diarrhea, malnutrition, etc), the village of 
residence, the person’s age and sex, diagnosis (e.g., malaria, pneumonia, etc), 
treatment (e.g., antibiotic, antimalarial, etc), and timing of the visit (e.g., first versus 
follow-up visit). Note that this data is already routinely collected on paper forms. 
Identifying information like names will not be collected. 
In each CSPS included in the Mother MUAC assessment, we will collect data on 
malnutrition including name of participants in the program, study ID, village of 
residence, date of admission in the program, measurements of weight and height, 
treatment received and outcome. Notes that this data is already collected on paper 
forms 
Passive surveillance in the district hospitals located in our study area: we will collect 
study ID, name of participant, village of residence, date of admission and discharge, 
reason for hospitalization, treatment, and outcome of hospitalization for all children 
hospitalized under 6 months of age. Notes that this data is already collected on paper 
forms 
 
 
7.1 
5.3 Individually randomized targeted treatment 
 
Recruitment 
There are three occasions in which children could be recruited for the targeted 
treatment:  


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1)During the 1st BCG visit at approximately 5 weeks of life if the child is at 
least 29 days old.  
2) During the 6 week (42 days) postnatal follow-up visit or any other well-
child visit happening during the age of 5 weeks to 12 weeks  
3) When children are attending their first EPI vaccine visit at approximately 
12 weeks of life (1st EPI vaccine visit).  
Enrollment  
The children will be enrolled for targeted treatment at the health center or during 
other health outreach in the community after obtaining written consent from at least 
one guardian. Enrollment will happen at the Nouna district, the K. Vigué district and 
Banfora districts 
The children enrolled have to be living in a participating study community and be 
aged 28 days to 12 weeks. 
Anthropometric Baseline assessments 
All children enrolled in the study will undergo anthropometry: we will measure 
height, weight and middle arm circumference of each child. 
Treatment 
In all communities, children attending local health posts or children present at 
community health outreach will have the opportunity to receive a dose of 
azithromycin or placebo. Children will be individually randomized to receive 
placebo or azithromycin. Receipt of treatment will be recorded on the child’s study 
card. This treatment will occur whether the targeted treatment occurs during other 
health outreach or when the caregiver seeks vaccination at the health post or when 
the child visits the health post for a well-child visit. Community health workers will 
conduct a household visit when the mother and child do not come to the health post. 
2-week infant adverse event survey 
To identify any adverse events associated with the individually treated children, 
the research team will perform an adverse event survey approximatively 2 weeks 
after the treatment has been administered in a random subset of children. 10% of 
the children treated as part of the study will be randomly selected and the 2-
week IAES will be performed. This survey will be performed by the census 
workers masked to treatment arm. A structured questionnaire will be performed 
to elicit adverse events following treatment, followed by an open-ended 
question. Specifically, we will ask the primary caregiver about the following 


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symptoms during the time since the previous antibiotic distribution: abdominal 
pain, vomiting, diarrhea, constipation, hemorrhoids or rash.  
 
Six-month Mortality 
All children treated in all study communities will be followed for 6 months for vital 
status assessment. At approximately 6 months of age, a field worker will assess the 
vital status of the child (alive, died, unknown) and their current residence (residing 
in the household, moved, unknown). This visit will be an in-person visit to the health 
post.  
Six-month Anthropometry assessment 
All children treated during the targeted treatment visit in all study communities will 
be followed for anthropometry assessments at approximatively 6 months of age. The 
child will be measured, weighted and we will measure the middle upper arm 
circumference. This procedure will be done at the health post during the 6-month old 
vaccine visit. 
Rectal swab collection 
During the 36 months visit in the mortality plus communities we will obtain one 
rectal swab from up to 10 children per community who participated in the targeted 
treatment. 
 
8 Chapter 6:  Census  
6.1. 
Census 
 
Census Team 
Census workers will be selected by the CRSN study coordinator. These 
individuals may have different qualifications and educational backgrounds, but, 
at a minimum, each census team member should be computer-literate, such that 
they are able to operate a tablet computer and type on its keyboard. In addition, 
several supervisors will be present for the duration of the census to monitor 
census workers. 
 
Census Training 
Census workers will be trained at the beginning of the study and refresher 
trainings will be offered as needed for the duration of the study. Training will 
start with reviewing the census data collection software on the tablet computer, 


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care of the tablets, charging of the tablets, etc. The training will then proceed to a 
demonstration of the use of the software at a mock household, including 
common problems that staff may encounter (e.g., no one at home, GPS function 
not working, software crashing). In the final part of the training, the study 
coordinators and investigators will accompany team members to several 
communities and observe the census activities.  
 
Census Software  
The census will be directly entered into a tablet computer. The software will 
capture information about each child aged 0-5 in each household: name, age, sex, 
father’s name, and mother’s name, and will also register any pregnant women. 
The GPS coordinates will be documented for each household at the entrance to 
the household. At follow-up censuses, team members will identify each 
household on the existing census, and will update the status for each child:  
- 
STATUS: Alive, slept in household last night 
- 
STATUS: Alive, but not in household 
o ABSENCE: <1 month 
▪ Is he/she coming back within 1 week? 
• Yes (mop-up) 
• No 
• I don’t know 
▪ INFORMANT: household member, neighbor, village chief, 
other 
o ABSENCE: ≥ 1 month 
▪ MOVE: Moved within community 
• INFORMANT: household member, neighbor, village 
chief, other 
▪ MOVE: Moved outside of community 
• INFORMANT: household member, neighbor, village 
chief, other 
- 
STATUS: Died 
o PLACE: Child living in community when died 
▪ INFORMANT: household member, neighbor, village chief, 
other 
o PLACE: Child had moved out of community when died 
▪ INFORMANT: household member, neighbor, village chief, 
other 
- 
STATUS: Unknown 
o INFORMANT: household member, neighbor, village chief, other 
  


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Whenever a new individual is added to the census, the software will 
automatically assign each individual to a universal unique identification number 
as well as a study identification number.  
 
Census Data Uploading 
The census will be collected on tablet computers with 3G mobile and Wi-Fi 
capabilities. There will be 3 options for uploading data to the database. First, and 
most desirable, a SIM card with data plan can be purchased for each tablet, and 
the data uploaded via cell towers once per day (at the end of the day). This 
option is most desirable because it minimizes data loss in the case of a lost, 
stolen, or damaged device. In addition, this option will not require each tablet 
computer to be in contact with a Wi-Fi hub. As a second option for uploading, 
each census supervisor will have access to a Wi-Fi hub, and the supervisor can 
visit the census teams to upload data regularly. This option is less desirable, 
because the data will be uploaded less frequently. As a third option, the data can 
be uploaded at a central study site, either via Wi-Fi or micro USB cable directly 
into a computer. This option is least desirable because is not feasible to take the 
tablet computers to the central site regularly given the large geographical areas 
of the study. 
 
Census Supervision  
The CRSN study coordinator will supervise all census activities. Formal checks 
of census quality will be conducted through the random census verification. In 
addition, the study coordinator and CRSN GIS team will visualize all censused 
households using imagery from GoogleMaps. The goal of this activity will be to 
minimize the chances of missing large neighborhoods or specific regions within 
study communities. The study coordinator will also check the data entry 
progress for each community, paying special attention at the follow-up censuses, 
as to whether there are any missing data for the “vital status” variable (i.e., 
present, dead, absent). Once the study coordinator is confident that the entire 
community has been reached, and that the amount of missing data are acceptable 
(defined as <10% of children in a community), the study coordinator (or other 
research team member) will certify the census data collection for that community 
complete via Salesforce. Changes can be made to the record at different time 
points, but these changes will not be reflected until the subsequent census. All 
changes are time and date stamped in the database.  
 
Census Timing 
The census will be performed prior to each mass azithromycin/placebo 
distribution. Study sites may choose to perform the census activities over a 
discrete time period (e.g., all communities completed over a 1-month period, 
requiring census activities to take place simultaneously in many communities at 


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once) or alternatively in a “rolling” fashion (e.g., all communities completed over 
a 6-month period, requiring fewer census teams to be active at once). In either 
case, each community must be censused every 6 months, so it may not take more 
than 6 months to complete all communities. The census must be completed (i.e., 
“locked”) at the household level before treatment can be given.   
 
6.2. 
Random Census Verification 
A random re-census of households will be conducted at each study visit by 
supervisors, additional census team members, or local community monitors, as 
appropriate for the study site. Verification will be performed at the household 
level, with a minimum of 200 households being resurveyed during the 7 study 
visits (approximately 30 per visit).  Each team must have at least one household 
census verified at each study visit.   
 
Households will be selected using a different mechanism than the mechanism 
used by the original census. The primary method for selecting random 
households will be from aerial visualization (Google maps, AfriPop, etc.) If aerial 
visualization is not possible in an area, then another method for obtaining 
households can be used, such as a random walk. 
 
Both census teams will use the same electronic template (i.e., no prior records at 
MORDOR 0, and the census records from the prior census at each follow-up 
visit). We will arbitrarily select a sample of communities stratified by census 
team. Once the census has been completed, the trial biostatistician will analyze 
the communities for verification.   
 
The trial biostatistician will compare the results of the original census and the re-
census to identify any discrepancies. The steering committee will determine any 
corrective actions once this comparison is made. At the very minimum, the site 
study coordinator will inform the original census team of the discrepancies and 
will conduct a refresher training session to minimize data collection errors.  
 
6.3. 
Verbal Autopsy 
Verbal autopsy questionnaires will be completed for all deceased children (aged 
1-60 months) in the whole Nouna district.   
 
Staff 
Verbal autopsy interviews will be conducted by trained staff. Training will focus 
on conducting sensitive interviews with persons who may still be in mourning; 
discussion of verbal autopsy questions; reviewing the format of the paper 
and/or electronic questionnaires (including skip logic); and demonstration of the 
verbal autopsy technique on 5 mock deaths. The 5 first verbal autopsies will be 


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observed by the study coordinator to ensure that proper procedures are 
followed. Each verbal autopsy interviewer will be responsible for a distinct 
geographic area, and will be responsible for regular contact with the key 
informant from each community.  
 
Identification of Deaths 
Deaths will be identified in 2 ways: from the biannual census, and from the key 
informant system. The CRSN data manager will provide a list of all deaths to the 
site study coordinator after each census. This list will include information on the 
deceased child’s name, age, gender, and unique identification number; 
community name; and father’s and mother’s names. The study coordinator will 
deliver this list to the appropriate verbal autopsy interviewer. The verbal 
autopsy interviewer will also keep a record of all deaths identified by the key 
informants. In each case, the key informant will report the community name, 
child’s name, and parents’ names, and the deceased child will be located in the 
census database. 
 
Questionnaire Administration 
The questionnaire will be administered as is done routinely for the HDSS. 9 The 
HDSS uses the WHO standard verbal autopsy questionnaire WHO-VA-2016 and 
cause of death will be assigned using InterVA 4.10 Questionnaires will be 
administered at the home of the deceased child. The informant will be the 
deceased child’s parent or guardian. If this person is not available, the verbal 
autopsy interviewer will try to arrange a time to return to interview this person. 
If the parent or guardian is not present on the third visit, they will complete the 
questionnaire by interviewing another family member, or as a last resort, a 
neighbor. All interviews will be completed in the local language. Our goal is to 
perform each verbal autopsy within 1 to 6  months of identification of death.  The 
child’s name and unique identification number will be recorded on the verbal 
autopsy record for identification purposes. 
 
Assigning the Cause of Death 
As recommended by the WHO, we will use automated methods to assign causes 
of death based on the verbal autopsy questionnaire, rather than physician 
review. We will treat all individuals under 4 weeks as one subpopulation, and 
individuals 1-60 months as a separate subpopulation for determination of cause-
specific mortality fractions.  
 
6.4. 
Validating the mortality outcome 
 
No death registries exist at the health facility. All Deaths from our census will be 
investigated and will catch false positive deaths recorded during the census. 


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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.   
We will record all deaths happening at the health facilities to be able to catch 
deaths the field workers might have missed during the census. 
The study coordinator will work closely with the health centers to be able to 
obtain a list of deaths occurring in his/her facility.  
 
The Data Manager will be responsible for linking the deaths occurring at the 
health facilities with the census file, using the name, age, and village of deceased 
children.  
 
Verbal autopsies will be performed on all deaths picked up by the census and 
also all deaths from 0-5 year-olds picked up by the health facility if not included 
on our census. 
 
Chapter 7: Registering Participants for Specimen Collection 
Samples will be collected with reference to age, gender, household, and 
community, but participant names will not be included in laboratory records to 
ensure privacy.  Samples will thus not be associated with an individual’s name, 
but with a random identification number and/or QR code, masking laboratory 
personnel and preventing identification of individuals.   
 
At each time point, each child selected for examination/specimen collection will 
be assigned an identification number for database anonymity.  
 
For each community, the randomized registration list for examinations will be 
generated by the database and downloaded to the tablet using the mobile 
application.  
 
After registration, the child and his/her guardian will be directed to the 
appropriate examination stations.  
 
Chapter 8: Blood samples  
We will collect:  


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1) Thick and thin smears, assessed for malaria parasitemia and 
gametocytemia by CRSN microbiologists, 
2) Microcuvettes, analyzed for hemoglobin in the field using a HemoCue 
analyzer (HemoCue AB, Ängelholm, Sweden), and 
3) Dried blood spots, collected on FTA Elute cards (Whatman, Kent, UK; or 
appropriate substitution) and sent for laboratory testing for malaria using 
a nested PCR assay40 and/or TropBio cards (Tropbio Pty Ltd, QLD, 
Australia) for serologic testing.  
 
The order of events at the blood collection station is: 1) finger prick; 2) blood 
spots on filter paper; 3) hemoglobin test; 4) thin and thick smears for malaria.  
 
 
 
8.1. 
Fingerstick 
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. 
 
A finger stick of capillary blood will be collected for thin and thick blood smears 
to assess for malaria, hemoglobin testing, and dried blood spots to be stored for 
later testing. 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.  
 
Fingerstick procedure: 
1. Prepare the disposable lancet. Use a NEW disposable lancet for each child. 
Do not re-use lancets!  
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. 


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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. 
 
8.2. 
Dried Blood Spots for Serology 
 
Collecting the FTA Elute filter paper sample: 
1. Label the filter paper with a random number sticker.  
2. Place 2-4 large drops of blood directly from the thumb or finger onto the 
large circle on the filter paper (if it is difficult to obtain 4 drops of blood, it 
is sufficient to collect 2 drops of blood).  
3. Leave the filter paper to air dry for a few minutes, then place the sample 
into a small plastic bag along with a desiccant packet.   
4. Leave the bag open for a few minutes more,  and when the blood is 
completely dry, roll down the top of the bag and close with a piece of 
masking tape. 
5. Store the filter paper samples (in small plastic bags) in a larger Ziploc bag. 
Keep all filter paper samples in a safe, dry place at room temperature. 
6. Blood spots will be stored at room temperature in a locked cabinet in the 
study coordinators’ office.  
 


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Collecting the TropBio filter paper sample: 
1. Label the filter paper with a random number sticker.  
2. Grip the filter paper on the side without small circles. Place a droplet 
of blood directly from the thumb or finger onto five of the six circles, 
leaving the right one blank. Be sure to fill each circle completely.  
 
 
 
 
 
 
 
 
 
 
 
 
3. The recorder will scan the QR code. 
4. Carefully slide the filter paper onto a pencil to air dry for at least an 
hour. There should be about 1 cm in between each sample. Secure the 
pencil into a Styrofoam surface in a box or container to protect from 
dust. 
5. When the filter paper is dry, place each sample into a small zip plastic 
bag (individually). Place the small bags into a larger Ziploc bag with 
five desiccant packets.  
6. Ensure the large Ziploc bag is sealed tightly, as moisture will damage 
the samples. Transport these filter paper samples to a freezer. 
Area to hold the filter paper.  
Do not touch the small circles. 
Leave last circle blank 


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Set up HemoCue Analyzer 
1) Remove the HemoCue analyzer from the case. If a small battery 
symbol appears on the top right side of the display, the batteries are 
low. The HemoCue will still give accurate results, but it is strongly 
recommended to replace the batteries as soon as possible. 
2) Pull the cuvette holder out to the loading position. Press and hold the 
left button until the display is activated (ALL symbols appear on 
display). The display will show the version number of the program, 
an hour-glass symbol and “Hb.” At this time, it will perform an 
automatic SELFTEST to verify the performance of the device. After 10 
seconds, the display will show three flashing dashes and the 
HemoCue symbol. This means that the HemoCue has passed the 
SELFTEST and is ready for use. If the SELFTEST fails, an error code 
will be displayed. 
 
8.1 
Set-up drying area for TropBio bloodspots 
Supplies: pencils, Styrofoam, cardboard box, paper 
- Place Styrofoam in cardboard box 
- Put pencils inbox/container – space apart 
- Note: When placing the blood spot samples on 
the pencil, space apart by ~2.5cm with pieces of 
paper in between each sample. 
 
 
 
 
 
8.3. 
Hemoglobin Test 
A portable spectrophotometer (HemoCue, Anglom, Sweden) will be used for 
hemoglobin testing.  
Collecting a blood sample for hemoglobin (HemoCue): 
1. Remove a cuvette from the container. Reseal the container immediately.  
(The recorder can help the examiner with this.) 
2. When the blood drop is large enough, fill the microcuvette in one 
continuous process. Do not refill! If there is not enough blood to fill the 
microcuvette, you must start again with a new microcuvette. Wipe any 
excess blood from the sides of the microcuvette with clean gauze or a 
paper towel, but be careful to avoid touching the open end of the 
microcuvette so blood is not removed. 
3. Look for any air bubbles in the filled microcuvette. If air bubbles are 
present, discard the microcuvette and obtain a new drop of blood using a 


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Cleaning the HemoCue Analyzer 
1) To clean, pull the cuvette holder to the loading position.   
2) Carefully press the small catch (upper right corner of the cuvette 
holder). Continue to press the catch and carefully rotate the cuvette 
holder as far left as possible, and then carefully pull the cuvette holder 
out of the analyzer.   
3) Clean the cuvette holder with alcohol or a mild detergent. Push a clean 
cotton tipped swab moistened with alcohol (without additive) into the 
opening of the cuvette holder and move from side to side 5 – 10 times. 
If the swab is dirty, repeat with a new clean swab until the cuvette 
holder is clean. A dirty cuvette holder may cause the HemoCue 
analyzer to display an error code. 
4) After 15 minutes (or less time, depending on the climate), you may 
replace the cuvette holder and use the analyzer. The cuvette holder 
must be completely dry before you replace it. 
5) Put the Hemocue analyzer back into its case. 
new microcuvette.  (Small bubbles around the edge of the microcuvette 
can be ignored.) 
4. Place the filled microcuvette in the cuvette holder. Gently slide the cuvette 
holder to the measuring position to be analyzed immediately. (This must 
be performed within 10 minutes after filling the microcuvette.) 
5. After 15 – 60 seconds, the hemoglobin value will be displayed. The 
examiner should read the hemoglobin value aloud so the recorder can 
enter it into the tablet computer. The value will remain on display as long 
as the cuvette holder is in the measuring position. The analyzer will turn 
off automatically after 5 minutes.   
Note: For children 6 months to 5 years, if the hemoglobin is <11.0 g/dL, 
the child is anemic. For children 5 – 11 years, if the hemoglobin is <11.5 
g/dL, the child is anemic (WHO/UNICEF/UNU, 1997). If a child is found 
to be severely anemic, the examiner must refer him/her to the nearest 
health center for treatment.  
6. Carefully dispose of the used microcuvette in the sharps container.   
7. At the end of the day: Turn off the HemoCue analyzer. Press and hold the 
left button until the display reads OFF. The display should be blank. 
8.4. 
Thick and Thin Smears for Malaria 
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.   


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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. 
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. 
 
A rapid diagnostic test (RDT) could be substituted for thick smears if approved 
by the Steering Committee.  
 
8.4 
Rapid Diagnostic Test 
A rapid diagnostic test for malaria will be conducted at the final exams (CHAT 36).  
Blood will be obtained from the finger prick site of the previous exams. A pipette will 
be filled with blood which will then be placed into the corresponding chamber on the 
test.  
Three to 6 drops of the buffer solution will be placed into the chamber provided on 
the test.  
The examiner will wait 15 minutes before reading the test.  
Negative: A single red line appears under the letter “C” on the test. This is the 
control.  


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Positive: 2 red lines appear. One line under “C” on the test and a second line under 
“P.f” indicating P. falciparum.  
If the result is positive, the child will be referred to the health center for treatment. 
 
8.5. 
Materials for Blood Collection 
 
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  
 
Dried Blood Spots 
FTA Elute cards 
Small zip plastic bags  
Desiccant packs  
Masking tape 
Large Ziploc bags (handful) 
TropBio circular cards 
Small zip plastic bags 
Desiccant packs 
Large Ziploc bags (handful) 
Materials for drying apparatus: 12 sharpened pencils, Styrofoam, empty 
cardboard box 
 
Hemoglobin Test  
HemoCue machine 
Extra set of AA batteries  
Cuvettes 
Q-tips (handful) 
 
Thick and Thin Blood Smears 
Glass slides  
Slide box 
 
Rolls of random number stickers 
 


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Rapid Diagnostic Test 
RDT 
Solution 
 
Google Nexus 7 
External battery pack 
9 Chapter 9: Specimen Collection for Resistance Testing  
 
9.1. 
 Population 
We will collect nasopharyngeal and stool samples on a random set of 15 children 
aged 1-60 months from each of the Mortality Plus communities. Children will be 
selected from the current study census. The swabbing visits will occur after the 
census but before treatment. The randomized registration list of children will be 
provided to the site study coordinator before the swabbing visit. The Study 
Coordinator will give this list to the community for mobilization prior to 
examinations. 
 
 
9.1 
9.2 Nasopharyngeal Swabs 
Nasopharyngeal swabs will be stored in DNA/RNA shield media by Zymo and 
STGG media, and standard microbiologic techniques will be used to isolate S. 
pneumoniae and test for resistance to azithromycin, penicillin, and clindamycin. 
Resistant isolates will be assessed for the most common genetic resistant 
determinants (ermB and mefA) using a PCR-based assay.41 Serotype will be 
assessed using a nested PCR reaction for the most common serotypes, followed 
by the Quellung reaction for any untyped isolates.42 
 
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 


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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. 
9.2.1 
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).  
 
 
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. 
 
-20°C Freezer 
A standard -20°C freezer located at the CRSN laboratory will be used strictly for 
the storage and freezing of ice packs and samples. This freezer is kept in a locked 
room on the grounds of the CRSN Laboratory, which is under 24-hour security 
guard supervision. 
 
-80°C Freezer 
A dedicated -80°C freezer located at the CRSN laboratory will be available for 
storage of study samples, including rectal and nasopharyngeal swabs. 
 
9.2.2 
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 
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. 


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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. 
Upon returning from the field each day, the samples in STGG will be 
immediately taken to the CRSN laboratory and stored in a commercial -20°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. 
 
9.3 Stool Samples  
All study sites will collect either stool samples or rectal swabs for resistance 
testing, but methodology will depend upon the type of samples collected and 
consist of either culturing/microbiological testing or nucleic acid based testing.   
 
Rectal Swabs for Culturing/Microbiological Testing  
Rectal swabs will be collected and placed into Amies transport media or Norgen 
Stool Preservative. We will use standard microbiologic techniques to isolate 
Escherichia coli. Resistance to azithromycin, ampicillin, and co-trimoxazole will be 
determined. Isolates can be further classified into commensal and diarrheagenic 
subtypes using a multiplex PCR assay.11 
 
Stool Samples for Nucleic Acid Based Testing 
Stool specimens will be used to look for the presence of E. coli and macrolide 
resistant determinants typically associated with resistant strains of E. coli, using 
a resistome approach. This will be carried out by isolating DNA from stool 
specimens and detecting the presence of E. coli as well as genes associated with 
antibiotic resistance (i.e. erm, mef, and mph genes) via PCR assays.  Other possible 
experiments may eventually include using PCR to detect the presence of toxins 
or virulence factors (i.e. eae, stx, bfp-A,VT-1, VT-2), which are linked to 
diarrheagenic E. coli strains, and exploring the complete microbiome of the stool 
specimens using DNA and RNA sequencing. 


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9.3.1 
Stool Specimen Collection 
 
Rectal Swab Collection for Culturing/Microbiological Testing  
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 temperature (7 days for RNA), and thereafter 
can be frozen (-20°C or -80°C) for long-term storage. 
 


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Stool Specimen Collection for Nucleic Acid Based Testing 
For the study participant/parent of the child: 
1. Collect the initial stool specimen on a piece of plastic.   
2. Transfer a few heaping spoonfuls of the fresh stool into the smaller, 4 oz 
disposable plastic container that has a locking lid, using the spoon 
provided. Return this to the trained field worker. 
 
For the trained field worker: 
1. Wearing fresh gloves, carefully place a portion of the stool sample from 
the disposable 4 oz plastic container into a labeled Norgen Stool Nucleic 
Acid Collection and Transport Tube (15 ml collection tube that contains 
preservative), using the small spatula that is attached to the tube’s cap. Fill 
up to the line as indicated by the tube.44 Make sure to spoon the stool into 
the tube without touching the rim or outside of the tube to avoid any 
contamination. 
2. Once the stool sample has been added, place the cap tightly back onto the 
tube. 
3. Mix gently until the stool is well submerged under the preservative. Do 
not shake the tube up and down, just gently swirl. 
4. Wrap the lid of the tube with a piece of Parafilm to seal it. 
5. Place the tube into the storage box and store at room temperature. 
6. Once the final stool sample has been collected in the Norgen Stool Nucleic 
Acid Collection and Transport Tube, wrap up the initial stool sample in 
the large receptacle container using the plastic lining and properly dispose 
of it. Also, dispose of the stool sample in the 4 oz plastic container and the 
spoon that was used. 
 
9.3.2 
Materials for Stool Specimen Collection 
 
Rectal Swab Collection for Culturing/Microbiological Testing  
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. 
 
Stool Specimen Collection for Nucleic Acid Based Testing 


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Plastic Lining 
Each participant will be given a piece of plastic that will be placed on the ground 
to collect the initial stool sample.  
 
Spoon 
Wooden medical spoon used to transfer a portion of the initial stool specimen to 
the small plastic container, which will be brought to the trained field worker by 
the parent. 
 
Small Plastic Container 
4 oz disposable plastic container with locking lid used to transport a portion of 
the initial stool specimen to the trained field worker. 
 
9.1.1 
9.3.3 Protocol for Fresh Frozen Stool Collection 
Materials 
• Stool specimen (10-20 grams or ml) 
• Pre-printed PID labels (4 plus 1 extra) 
• 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)  
Collection Procedure  
The stool sample is to be collected within a two day window of the scheduled 
time. Even with the best of efforts the field worker fails to collect stool sample 
within 2 days window, field worker may visit the home up to 5 days beyond that 
+2 time frame.  
For collection of Stool, inform child’s/participant’s primary caretaker/ 
participant one day before planned stool collection and request caretaker to 
collect the first available fresh stool sample from the child on the morning of the 
planned visit. 


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The mother / participant’s primary caretaker/ participant should be provided 
with the labeled stool container, diaper (for infants), cold box, ice packs, gloves, 
plastic spoon, and 2 plastic bags the evening before planned stool collection. 
There should be enough ice packs in the cold box to keep it cold for up to 8 
hours.   
Instruct the caretaker/ participant to 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.   
Temporary Storage and Transport Procedures 
 
Instruct the caretaker to place the plastic bag with the stool in the cold box 
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) 
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. 
 
Collection and Transport Procedures for Microbiome analysis  
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].  


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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. 
 
9.4 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.  
 
Duplicate swabs 
Duplicate NP swabs and rectal swabs/stool specimen will be collected from two 
children per community.  
 
 
10 Chapter 10: Training  
 
10.1 
Standardization 
The research team will work together prior to the baseline visit to standardize all 
study procedures. We will review the format, general logistics, and procedures for 
the house-to-house census. The importance of capturing the vital status of every 
individual in the study area, including individuals not on the previous census (i.e., 
new births, deaths and migrations) will be stressed. The importance of capturing 
those individuals who were born and died in the time period between two censuses 
will also be highlighted. Census workers who have successfully completed the 
training will be certified, although certification can be revoked on subsequent quality 
control checks. Ongoing training activities (before each biannual census) should 
consist of didactic classroom instruction and mock census activities, followed by in-
field training, reviewing the use of the electronic data capture, including charging 
devices and troubleshooting technical problems. 
 
Chapter 11: Sample Organization, Transport, and Storage 
 


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11.1 
De-identification 
All specimens will be labeled in the field with a random identification number 
linked to the census in the electronic data capture system, but to facilitate 
masking, only the CRSN DCC will have access to the key linking the ID with 
census information. Age, gender, and community of residence will be available 
for each specimen, but names will be kept confidential. Therefore, all specimens 
will be de-identified. 
 
11.2 
Specimen Transport  
After sample collection, samples from the field will be transported to the CRSN 
laboratory for storage and processing. 
 
During any international specimen transport, the temperature of the shipper 
boxes will be documented by a temperature recording device. 
 
Blood Samples 
Blood smears (thin and thick) will be transported at room temperature to the 
CRSN laboratory. FTA Elute cards will be transported at room temperature and 
stored at the health clinic before being transported for processing. TropBio cards 
will be transferred on ice to the health center and stored at -20°C prior to 
shipment.   
 
Swabs 
Swabs in STGG media will be initially stored in the field at -4°C using a closed, 
insulated container until arrival at a securely locked freezer at -20°C. Swabs in 
DNA/RNA Shield media will be stored in ambient temperature in the field and 
then transferred to a refrigerator or freezer.  
 
Stool Samples 
Rectal Swabs for Culturing/Microbiological Testing  
Rectal swabs preserved in Amies transport medium should be refrigerated until 
processed. If specimens will be kept more than 2 to 3 days before being cultured, 
it is preferable to freeze them immediately at -80°C. It may be possible to recover 
pathogens from refrigerated specimens up to 7 days after collection; however, 
the yield decreases after the first 1 or 2 days. Frozen specimens should be 
transported on dry ice. 
 
Stool Samples for Nucleic Acid Based Testing  
Specimens preserved in Norgen Stool Nucleic Acid Collection and Transport 
Tubes can be left at room temperature for 7 days (if preserving RNA) or up to 5 
months (if preserving DNA). Specimens can also be transported in the 


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preservative at room temperature. If the samples will be kept for long term 
storage, they can be placed in a -20°C or -80°C freezer.  
 
11.3 
Specimen Storage 
Sample storage will occur in two stages: short-term and long-term. 
 
11.3.1 Short-term Sample Storage 
Samples will be labeled with study ID only and are unidentifiable without access 
to the study database. All samples will be transported to the CRSN laboratory for 
storage and processing. A subset of samples will be shipped to UCSF to process 
core samples and any secondary processing. 
 
11.3.2 Long-term Sample Storage 
All samples processed by the CRSN laboratory will be stored in the -80°C freezer 
for at least 5 years. A subset of de-identified samples from Burkina Faso will be 
shipped to UCSF for longer-term storage at the UCSF Oyster Point Facility, 
which is designed particularly for secure long-term (5 years) storage of biological 
specimens, at -80°C for future analyses by CRSN and UCSF investigators and 
other interested parties.     
 
11.4 
Catalog Specimens 
We will create a list of study data and specimens, including the age, gender, 
village identification number, treatment assignment, whether treatment was 
received, vaccination record, and symptom questionnaire. We will also list the 
date of collection and transport, and the storage conditions while in the field and 
while banked at UCSF. This will facilitate identification of specimens for future 
analyses. 
 
11 Chapter 12: Study Medication 
Children aged 1-59 months on the current census will be offered weight- or 
height-based, directly observed, oral suspension (azithromycin or placebo) every 
6 months for 3 years (as performed in trachoma programs) at each study site. At 
the final phase of CHAT, all children 1-59 months will be offered azithromycin. 
Children under the age of 12 months or not able to stand will be weighted. In 
addition to being at least 1 month of age, children should weigh at least 3.8 kg to 
be eligible for treatment. This ensures that mistakenly aged or premature infants 
won't be treated. These infants will be eligible for treatment at the subsequent 
distribution, approximately 6 months later. The mortality application will not 
provide a dose for children weighing <3.8 kg. 
 


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We will monitor adverse events following mass treatments as described in the 
adverse events section. The treatment and monitoring schedule for all study 
arms is shown in Table 1. 
 
11.1 12.1 Study Medication Description (from Pfizer, Inc.) 
 
Azithromycin 
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. 
 
12.2 
Dosage Information 
Azithromycin and placebo will be administered as a single dose, in oral 
suspension form for children. 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)  
• Height-based dosing of children ( this dosing method is supported by 
the WHO) 
 
Individuals who are allergic to macrolides/azalides will not be treated. 
 
12.3 
Medication Procurement/Donation 
Azithromycin (Zithromax®) and the placebo have been donated by the Pfizer 
Corporation.  There will be no costs to acquiring the study medication. Pfizer, 
Inc. will ship azithromycin and placebo directly to the study sites. 
Representatives of the study site will manage the customs process and transport 
the medication from the port to storage sites.   
 
12.4 
Medication Quality Control 
Study medication will be shipped directly from Pfizer and stored at CRSN prior 
to use. The study coordinator and other staff will regularly check and record the 
study medication expiration dates. We will strictly monitor expiration dates on 
the medication containers and all expired study medicine will be discarded 
appropriately. 
 
12.5 
Antibiotic Distribution & Monitoring Coverage 
After the MORDOR 0 census and monitoring/collection is complete, treatment 
(azithromycin and placebo) will be administered to all eligible community 


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members per study protocol. Teams will participate in training exercises 
regarding drug/placebo distribution and recording techniques prior to each 
treatment cycle. Training will be in accordance with the Zithromax Program 
Manager’s Guide from the International Trachoma Initiative.    
 
During mass drug administration, distribution team members will use tablet 
computers equipped with an electronic data capture system to seek out each 
eligible child on the census, administer antibiotic or placebo, and record whether 
or not each person has been treated.  The distribution team will document 
individual reasons for not being treated (e.g. death, temporary absence, 
permanent migration, refusal of treatment, etc.). Consumption of medication will 
be directly observed and the dose distributed will be documented in the 
electronic data capture system. 
 
We will estimate antibiotic coverage from the most recent biannual census 
records, aiming for treatment of 80% of children. At the end of each treatment 
round, the DCC will identify any children who have missed 2 or more 
consecutive treatments, and relay this information to the study coordinator. 
Census teams will discern the reason for missing treatments (including 
unrecorded death) at the next scheduled census. This system will serve as a 
quality control mechanism to reduce the number of false negative deaths in the 
study. 
 
12.6 
Adverse Reactions/Side Effects 
Azithromycin is generally well-tolerated.  The most common side effects of 
azithromycin are diarrhea, nausea, abdominal pain, and vomiting, each of which 
may occur in fewer than one in twenty persons who receive azithromycin. Rarer 
side effects include abnormal liver function tests, allergic reactions, and 
nervousness.  Diarrhea due to Clostridium difficile has been reported in rare cases.  
 
During the consent process, the common adverse reactions that may occur will 
be explained to parents/guardians and they will be advised to communicate 
adverse events to CRSN study staff immediately. If, for any reason, the 
participant needs further care, they will be referred to the nearest health center 
for examination and treatment. 
 
The trial sites will be masked to outcomes, so the responsibility for monitoring 
interim analysis will fall on the DSMC. Statistical monitoring is discussed in the 
Statistical Analysis Plan. The Data Safety and Monitoring Committee (DSMC) 
will be given authority to discontinue treatments at any time if there is evidence 
of unexpected harm.  
 


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12.7 
Adverse Events Systems 
Both active and passive monitoring systems for adverse events are in place for 
this study, and these monitoring activities will specifically include (but will not 
be limited to) treated 1-6 month olds. We will monitor adverse events following 
mass treatments actively at each follow-up census and during a house-to-house 
survey of all 1-6 month olds in a random selection of azithromycin and control 
communities.  
 
12.7.1 Passive Adverse Events Monitoring  
We will implement a passive monitoring system during the treatment phase, by 
instructing parents to report any adverse events in the two weeks following each 
mass azithromycin distribution to a local healthcare provider. Children will be 
referred for follow up care on a case-by-case basis.  
 
12.7.2 Active Adverse Events Monitoring 
 
 


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Infant Adverse Events Survey 
To identify any adverse events associated with mass treatment, the research team 
will randomly select 48 study communities (12 per arm) to participate in an 
adverse events survey. This survey will be performed by the census workers 
masked to treatment arm, approximately 2 weeks after a mass medication 
distribution during the first phase only (CHAT 0). During the survey, adverse 
events will be elicited only for study participants aged 1-6 months at the 
previous census. A structured questionnaire will be performed to elicit 
dangerous side effects, followed by an open-ended question. Specifically, we will 
ask the primary caregiver about the following symptoms during the time since 
the previous antibiotic distribution: abdominal pain, vomiting, diarrhea, 
constipation, hemorrhoids or rash. We will only collect this infant adverse events 
survey for the first phase. The rest of the phases of the trial will not collect this 
information.  
 
Training 
The household survey team will be the same individuals who conducted the 
census. They will be trained in survey administration methods, including: 
1. Obtaining informed consent  
2. Accurately selecting the appropriate households to interview  
3. Remaining neutral when asking questions (i.e. asking the question 
exactly as it is written on the paper in a neutral tone of voice, so as not 
to lead the respondent or introduce bias)  
 
Serious Adverse Events 
Any serious adverse events (SAE) will be reported to Pfizer. An IIR SAE Form 
(Investigator-Initiated Research Serious Adverse Events Form) will be completed for 
each event. (See Appendix for form and complete instructions.) 
 
According to Pfizer, an SAE is any adverse event that: 
• Results in death 
• Is life-threatening (i.e., causes an immediate risk of death) 
• Requires inpatient hospitalization or prolongation of existing 
hospitalization 
• Results in persistent or significant disability or incapacity  
• Results in a congenital anomaly or birth defect 
 
 
Or that is considered to be: 
 
• An important medical event 
 


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All community residents will be advised to alert a village health worker if they 
experience, within one week of mass treatment, a serious adverse event (by the 
preceding definition). An SAE report must be submitted for all deaths in the 
study – regardless of the time of treatment. The local health worker will report to 
the study coordinator; who must, within 24 hours, submit a Pfizer IIR SAE Form 
to mordor.burkina.sae@gmail.com. AS and TL will review, and forward to Pfizer 
and/or the Medical Monitor, as appropriate. SAEs must be submitted to Pfizer 
within 24 hours of receipt from the on-site coordinator. AS and TL will also 
forward SAE to DSMC if meets criteria of being possibly related to study drug. 
The reporting of any serious adverse event will follow national procedures in 
Burkina Faso.  
• In the event of serious events, the CSPS will contact the study doctors 
on the same day. The patient will then be evacuated to an appropriate 
level of care for management. 
• The declaration will be made to the National Agency for 
Pharmaceutical Regulation in accordance with the regulations and 
within the deadlines (7 days in the event of death or life-threatening 
prognosis, 15 days in other serious and unexpected cases, 15 days in 
new facts) at pharmacovigilance.burkina@sante.gov.bf   
• The ethics committees will also be informed of the occurrence of this 
event within the same time frame. 
 
Deaths that are reported to the study team outside of the biannual census 
(primary outcome) will be reported as an SAE to Pfizer. Note that deaths 
identified via the biannual census, which constitute the primary outcome, will 
not be reported as SAEs. Deaths that are reported to the study team as part of the 
biannual census will be reported to Pfizer in aggregate, not by arm, on a 
quarterly basis. 
 
 
12.7.3 Adverse Events Data 
We will keep records and report all adverse events of azithromycin to the DSMC. 
We will report both efficacy and side effects of azithromycin separately for the 1- 
6 month old age group. For any “sudden deaths” believed to be associated with 
azithromycin treatment, key informants will immediately notify the verbal 
autopsy interviewer via SMS message or another appropriate form of rapid 
communication. Reporting of non-serious adverse events will follow national 
procedures in Burkina Faso, as well. In the event of a non-serious adverse event, 
the CSPS will process the cases and the national reporting form will be 
completed. Any adverse event occurring during the trial will be covered by the 
study free of charge.  
 


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11.2 12.8  Supply issues  
If a study site runs out of a treatment letter, a request should be sent to 
mordor.burkina.tx@gmail.com to request a replacement for the community in 
question. This is not blanket permission to substitute one letter for another – if 
there are several communities for which the assigned treatment has run out, a 
separate request must be made for each community. 
 
The study site coordinator will make the request; TCP will determine the 
replacement letter, a member of the DCC will make the change(s) in the 
database.  
 
The field team must log out of the MORDOR mobile app and log in again for the 
changes to take effect on the front end. The replacement treatment letter will then 
appear in the app.  


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12 Chapter 13: Protection of Human Subjects 
Before the study begins, the research team will obtain formal ethical approval 
from their respective ethics committees as well as national ethical approval in 
Burkina Faso. In addition, staff will approach community leaders to describe the 
study and answer any questions. Study staff will proceed only if local leadership 
consents to participate. Verbal consent will be collected from the village 
leadership.  We will also obtain verbal consent from the head of household to be 
able to perform the census. To be able to examine and treat the children living in 
the household, we will obtain written informed consent from a parent or 
guardian. This written consent will contain information regarding all study 
activities with patient contact: examinations, and treatments.  We will collect one 
written consent form per child the first time we enroll the child. The subsequent 
visits we will explain the study to the parent/guardian of the child but we will 
only obtain verbal consent. Children will be included in the study only following 
the receipt of the written consent from a 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. Individuals who withdraw will be offered the same medical 
treatment outside the study. 
 
Children with wasting, stunting, malaria, or anemia will be referred for 
appropriate treatment by trained study personnel, at the nearest health center. 
 
13.1 
Institutional Review Board Approval 
 
 
UCSF Committee on Human Research 
UCSF’s Committee on Human Research will annually review study protocol for 
ethical approval. 
 
CRSN Comité Institutionnel d’Ethique  
The study protocol will be reviewed and granted ethical approval by the Comité 
Institutionnel d’Ethique at the CRSN headquareters before any patient-related 
research activities begin. 
 
National Health Ethics Committee of Burkina Faso. 
The study protocol will be reviewed and granted ethical approval by the 
National Health Ethics Committee of Burkina Faso before any patient-related 
research activities begin and annually. 
 


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13.2 
Informed Consent 
First, the chairman of each village will be asked for permission to include the 
village in the study.  Additionally, the study will be discussed with all adults in 
the village by team members who speak the local language(s). 
 
 
Informed consent scripts will be translated into local languages before the study 
can begin. Consent scripts will then be back-translated by a different party to 
ensure comprehension. Consent scripts will be submitted and approved by 
national IRB committees in Burkina Faso prior to study implementation. Then 
they will be read aloud to each study participant (and his/her parent/guardian) 
by a team member who is a native speaker of the local language to ensure that 
they understand the risks and benefits of participating in all study activities. 
Young adults and children under 18 years of age, who cannot give consent by 
law, will be included in the study only following the receipt of written informed 
consent from a parent or guardian. If, at any time, a parent or guardian elects to 
withdraw themselves or a family member from the study, it will be made clear 
that they will still be eligible for treatment.  
 
13.3 
Risks and Benefits of Study Procedures 
 
13.3.1 Verbal Autopsy 
As verbal autopsy requires a family member to answer questions about a 
deceased loved one, he or she might experience emotional stress and grief related 
to the death of the child. Interviewers will be trained to address these situations 
appropriately with awareness of the cultural context before they are allowed to 
conduct these verbal autopsies. If the family member is in need of a mental 
health intervention, referrals will be made by the interviewer. 
   
13.3.2 Swabbing Procedures 
There are minimal risks to the participant who receives nasopharyngeal, and 
nares 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. 
  
13.3.3 Stool Collection 
Stool samples have been collected in this setting before, with essentially no risk 
to participants. 
  


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13.3.4 Blood Testing 
Blood testing will include a pin prick to the finger or heel.  The major risk of this 
procedure is infection at the puncture site, though using aseptic technique will 
minimize this occurrence. Individuals in these communities are familiar with this 
procedure because all children who present at a health center with fever are 
offered the pinprick for a malaria thick smear. 
  
13.3.5 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, stunting, malaria, or anemia 
will be referred for appropriate treatment at the nearest health center. 


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13 Chapter 14: Study Monitoring  
The project will be continuously monitored by the supervisory team, which will 
consist of members from CRSN and UCSF. The supervisory team will conduct 
regular monitoring visits to study site locations, with UCSF team members 
accompanying CRSN team members at least biannually.  
14 Chapter 15: Data and Safety Monitoring Committee Charter 
This Charter is for the Data Safety and Monitoring Committee (DSMC) for 
Mortality Reduction after Oral Azithromycin II Burkina Faso (MORDOR II Burkina): 
OPP1187628.  
 
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. 
 
15.1 
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.  


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15.2 
DSMC Membership 
The DSMC is an independent multidisciplinary group consisting of 
epidemiologists, biostatisticians, bioethicists, and clinicians that collectively has 
experience in the management of infectious diseases and in the conduct and 
monitoring of randomized clinical trials including subsaharan Africa. 
 
15.3 
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 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.   
 
15.4 
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 


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current version of the case report forms. At subsequent meetings, committee 
members will receive Open and Closed Data Reports. 
 
Formal Interim Analysis Meetings 
One or more ‘Formal Interim Analysis’ meetings will be held to review data 
relating to treatment safety and efficacy, and quality of trial conduct. There will 
be at least two interim decisions to be made by the DSMC, at approximately 12 
months and 24 months into the study. 
 
15.5 
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.  
 
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.  
 


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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. 
 
15.6 
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 relatively short, 
2-year study. 


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15.7 
DSMC Contact Information  
 
Table 5: DSMC Contact Information 
Allen Hightower, Chair 
awh1953@gmail.com  
Amza Abdou 
dr.amzaabdou@gmail.com  
Jackie Glover 
Jackie.Glover@ucdenver.edu  
Wafaie Fawzi 
mina@hsph.harvard.edu  
Miriam Laufer 
mlaufer@som.umaryland.edu  


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15 Chapter 16: Data Collection, Management, and Security 
 
16.1 
Scope of Data 
Mortality and morbidity data will be collected in this trial. Mortality data includes: 
census, mortality, and treatment. Morbidity and resistance data includes the 
following: census, mortality, treatment, and morbidity assessments.  
Mortality Data 
Trained census workers will collect census data on all households in the study sites 
(name, birthdate, age, gender of all household members) and keep track of births, 
deaths, and migration of children eligible for treatment. In addition to biannual 
census updates, trained community health workers and study supervisors will 
conduct WHO verbal autopsy interviews through the duration of the study to 
provide information on the cause of death. Trained distribution teams will collect 
data on treatment status and dose given to all study participants, if treatment is 
provided apart from the time of census.    
 
Mortality-Plus Data 
Trained health workers will collect data on core morbidity assessments such as blood 
samples (thick smears and dried blood spots for malaria, microcuvettes for 
hemoglobin), stool samples, and nasopharyngeal swabs. Note that for de-
identification purposes a random number sticker will be affixed to each specimen 
collected. In addition, before sample collection, parents or guardians will be asked a 
standardized series of questions to determine whether the child has had recent fever, 
cough, or diarrhea. Clinic-based case finding will be conducted at local health clinics, 
which will involve transcription of health records.  
 
Certain morbidity assessments will be entered into handheld mobile devices at the 
time of the examination (e.g. hemoglobin, responses to symptom questionnaire), 
while lab results for thick smears, dried blood spots, nasal, nasopharyngeal, and stool 
specimens will be entered after confirmation. 
16.2 
Data Storage, Management, and Security 
Data will be recorded electronically using handheld mobile devices with custom-
made software applications and uploaded daily onto a secure, password 
protected, central server. Rapid transfer of electronically captured data will allow 
nearly real-time monitoring of activity at the study site. All handheld devices 
and data entry coordinating centers will be password protected, and all changes 
in data will be noted, including the date of the change, and the person who made 
the change. To ensure the quality of the data, we will conduct training sessions 
before each biannual census where needed. The central database application will 


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use hard disk encryption and physical protection of the server (which is to be 
maintained in a locked room accessible only to authorized personnel). The 
database will be based on mySQL (which supports standard SQL queries). Data 
will be backed up off site (providing integrity in case of the physical loss of the 
server). Data will never be deleted from mobile capture devices until at least one 
offsite backup has been completed. Data security during electronic transfer will 
be achieved through use of the Advanced Encryption Standard (AES). 
 
16.3 
Data Monitoring and Cleaning  
Data monitoring and cleaning will be overseen by the data coordinating center 
(DCC) at the coordinating site. Data collection will be monitored on a weekly 
basis by the site study coordinator using the dashboard function on Survey 
solutions. The survey solution dashboard will consist of the following reports by 
study site: Date Household Census Completed, Number of Households Census 
Completed by Village, Percent Household Census Completed by village, 
Treatment Status by Worker, Age Distribution by Worker, Sex Distribution by 
Worker, GPS Missing by Worker, GPS Missing by Village, Number of Records 
Synced by Date, Assigned Treatment by Given Treatment, Treatment Status by 
Age, Treatment Status by Village, Age Distribution by Village, and Sex 
Distribution by Village. 
 
The DCC will ensure that the site study coordinators log on to Survey solution 
weekly to confirm the status of the dashboard. In addition, upon each village 
census completion, the DCC will create and maintain a Stata program to identify 
data quality concerns. Any such concerns which must be addressed at the site 
specific level will be queried by the DCC. At every phase, as each village is 
completed and the data is considered cleaned, the data will be locked and a list 
of deaths will be generated and provided to each site for verbal autopsy.  
 


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16 Appendix 
 
Appendix 1. SAP 
Appendix 2. Infant Adverse Events survey 
Appendix 3. Pfizer Investigator Initiated Research Serious Adverse Event Report 
Form and Completion Guide 
Appendix 4. Lab Protocol  
Appendix 5. Community study forms 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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References  
 
1 
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. 
2 
MORDOR Study Group. Mortality Reduction After Oral Azithromycin 
(MORDOR) Study. 2017. 
3 
Sie A, Louis VR, Gbangou A, et al. The Health and Demographic 
Surveillance System (HDSS) in Nouna, Burkina Faso, 1993–2007. Global 
Health Action 2010; 3: 1–10. 
4 
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. 
5 
Golding N, Burstein R, Longbottom J, et al. Mapping under-5 and neonatal 
mortality in Africa, 2000–15: a baseline analysis for the Sustainable 
Development Goals. The Lancet 2017; : 1–12. 
6 
Muller O, Garenne M, Kouyate B, Becher H. The association between 
protein–energy malnutrition, malaria morbidity and all-cause mortality in 
West African children. Tropical Medicine & International Health 2003; 8: 507–
11. 
7 
Becher H, Muller O, Dambach P, et al. Decreasing child mortality, spatial 
clustering and decreasing disparity in North-Western Burkina Faso. Tropical 
Medicine & International Health 2016; 21: 546–55. 
8 
World Health Organization,, UNICEF. WHO/UNICEF Joint Statement: 
Managing possible serious bacterial infection in young infants 0–59 days old 
when referral is not feasible. 2017. 
9 
Yé M, Diboulo E, Niamba L, et al. An improved method for physician-
certified verbal autopsy reduces the rate of discrepancy: experiences in the 
Nouna Health and Demographic Surveillance Site (NHDSS), Burkina Faso. 
Population Health Metrics 2011; 9: 34. 
10 Byass P, Herbst K, Fottrell E, et al. Comparing verbal autopsy cause of death 
findings as determined by physician coding and probabilistic modeling: a 
public health analysis of 54 000 deaths in Africa and Asia. Journal of Global 


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Health 2015; 5: 1–9. 
11 Tobias J, Vutukuru S-R. Simple and rapid multiplex PCR for identification of 
the main human diarrheagenic Escherichia coli. Microbiological Research 2012; 
167: 564–70. 
 

SAP_001.pdf: 
CHAT:
A community-randomized trial
February 13, 2023
Version 1.6
Statistical Analysis Plan
UCSF Francis I. Proctor Foundation
Centre de Recherche en Sante de Nouna
University of Heidelberg
1


Introduction
This document (Statistical Analysis Plan, SAP) describes the planned analysis and re-
porting for the clinical trial, Azithromycin for the prevention of post-neotanal child-
hood mortality in Burkina Faso: a community-randomized trial. It includes specifi-
cations for the statistical analyses and tables to be prepared for the interim and final Clinical
Study Report. This study is designed 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 prevention of infant and child mortality in Burkina Faso: a
community-randomized trial, Manual of Operations
• Azithromycin for the Prevention of Neonatal, Infant, and Child Mortality in Burkina
Faso and Safety Assessment, Proposal
• 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. Ex-
ploratory 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.
This document will be reviewed prior to the enrollment of patients.
All subsequent
changes will be indicated by detailed change log in the Appendix.
2


Contents
1
Summary
4
1.1
Mortality Trial
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
2
Statistical Analysis
4
2.1
Planned Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
2.1.1
Mortality Trial
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
2.2
Interim Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
3
Sample Size Considerations
8
4
Randomization
10
5
Abbreviations
11
6
Revision History
12
3


1
Summary
1.1
Mortality Trial
The trial profile is given in the Manual of Operations. In the mortality trial, communities
are randomized to two arms: Azithro and Placebo.
The trial is a placebo-controlled,
double-masked (i.e., double-blind), community-randomized, clinical trial.
Objective.
Determine the efficacy of biannual mass azithromycin distribution versus
placebo in children aged 1–59 months on all-cause mortality.
We hypothesize that bian-
nual distribution of azithromycin will lead to significantly reduced all-cause mortality among
children aged 1–59 months after 36 months of treatment.
2
Statistical Analysis
2.1
Planned Analyses
2.1.1
Mortality Trial
The primary prespecified analysis will use the number of deaths in each community as
the outcome variable. For analysis, a death is counted when an individual in the target age
range 1–59 months is enumerated as alive on census i (i = 0, 1, 2, 3) and is not on census
i + 1 due to death. Census i takes place at the beginning of the study, and each subsequent
census takes place approximately six months later.
Primary Analysis.
The primary analysis will be conducted as negative binomial regression. The number of
person-years (for persons in the target age range) is derived by summing the person-times for
each period. The community-level predictor consists of treatment arm for the community.
Specifically, the treatment variable is a community level variable, not an individual level
variable.
The statistical significance assessed by permutation testing at the level of the
randomization unit.
4


Individuals who are in the target age range at the beginning of any study period, but
who are no longer in the target age range by the end, are considered to be fully enrolled and
contribute the full amount of person-time for that period.
The analysis will be two-sided, with a type I error rate (alpha) of 0.05.
Statistical considerations.
• Statistical tests will be conducted with Monte Carlo permutation based on the ran-
domization unit. The number of replications will be 10,000, unless the Monte Carlo
confidence interval for the P-value includes 0.05. In this case, 10,000,000 will be used,
and this fact reported.
• Tests for interaction by age sub-groups and by phase on the additive scale will be
conducted to evaluate for statistically significant subgroup differences.
• 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.
• For negative binomial regression, an aggregation parameter of 30 or greater will cause
us to re-fit using Poisson regression.
• 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.
• Individual level missing data (due to loss to follow-up or dropouts) is expected. If an
individual is lost to follow-up or otherwise unavailable during an interval of length D,
then the individual will not be assigned any amount of person time. If an individual
dies within an interval of length D, then the individual is assumed to contribute the
amount of person-time D/2 to the denominator (midpoint imputation).
• Village-level missing data could occur for various reasons, including field error, with-
drawal of support from village leadership, or other unforseen reasons. The analytic
5


plan will be to analyze all available information (including data from any community
prior to the time of dropout of that community). If a community drops out and then
re-enters, all available data (before and after the dropout period) will be analyzed.
Supplementary analyses.
The purpose of the supplementary analyses reported in this section is to assess the role
of statistical choices and data quality choices in shaping the result.
survival Cox proportional hazard regression of time to death, with inference based on
permutation test of randomization unit
Secondary analyses of mortality.
All secondary analyses will be sharply distinguished from the primary prespecified anal-
ysis and will be identified as such. Secondary analyses include outcome variables or planned
subsets which contribute either additional insight or address different scientific questions
than the primary analysis.
age Poisson regression at the individual level, clustering on community, including age as a
covariate (we will both adjust for age, as well as examine age-treatment interactions)
age Negative binomial regression, reporting using the same age categories as in MORDOR
Niger/Malawi/Tanzania (1–5 months, 6–11 months, 12–23 months, 24–59 months)
timing Cramer–von Mises comparison of death time conditional on mortality, between arms;
comparison of death time density between arms
the number of azithromycin doses received Logistic regression at the individual level,
clustering on community, using the number of azithromycin doses as the primary pre-
dictor
factorial analysis Logistic regression at the individual level, clustering on community, us-
ing the interaction term for the two factors, the community and individual arms
6


Additional secondary outcomes.
nutritional status Weight, height, and MUAC will be analyzed. MUAC will be compared
between arms using clustered linear regression (clustered on community). MUAC will
be available in all 341 communities; weight and height will only be available in specific
prespecified communities which are located in the HDSS.
malaria Malaria parasitemia is assessed using thick smears. The available data will be the
fraction of the sampled children who are positive. This is a longitudinally measured
fraction, and we propose modeling this using clustered linear regression on square-root
transformed data (with clustered logistic regression as a sensitivity analysis). These
data are only available in the 48 HDSS communities.
antibiotic resistance Like malaria parasitemia, phenotypic antibiotic resistance is a binary
outcome at the individual level, and a fraction at the community level. The same
analytic template as for malaria parasitemia will be used.
microbiome Intestinal and nasopharyngeal microbiome will be assessed. We propose to
examine L2-norm (Euclidean) distance using PERMANOVA with permutations at the
level of the randomization unit. We will also also examine L1 and diversities (L2 and
L1, or Simpson’s and Shannon’s). This outcome, like malaria and antibiotic resistance,
is only available in the 48 communities in the HDSS.
cause of death A verbal autopsy questionnaire for children will be used to collect data for
verbal autopsies[?]; details are provided in the MOP. Cause of death will be assigned
according to an algorithm based on a published verbal autopsy hierarchy. Cause of
death will be compared with the use of the chi-square statistic, with clustering taken
into account by community-level, using a permutation test to obtain the p-value.
2.2
Interim Monitoring
Efficacy.
The trial is designed specifically to collect three years of data, because of
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interest in whether the treatment effect is durable after the first. However, in case of a large
effect found after one year, it may be desirable to expand treatment to all communities.
A single interim analysis conducted at alpha of 0.001 will be conducted. Specifically, the
interim analysis will be conducted when mortality data are available for the first year (two
study periods).
The final analysis would be conducted at alpha 0.049.
Futility. An interim analysis of futility is proposed, to be finalized in consultation with
the Data and Safety Monitoring Board. Specifically, we propose to conduct negative binomial
regression on village-level mortality counts comparing the treatment and control arms (using
a common aggregation parameter) using only data from the first year. This analysis will
be conducted using simulation. We suggest consideration of the conditional power to detect
a 30% effect. If this drops below 20% at the interim analysis, discontinuation of the trial
or other changes to the protocol may be made in consultation with the Data and Safety
Monitoring Board.
Interim analysis will be executed by the trial biostatistician at the central site.
3
Sample Size Considerations
We use the following formula (cited from [?]):
c = 1 +
zα/2 + zβ
2 (λ0 + λ1)/y + k2(λ2
0 + λ2
1)
(λ0 −λ1)2
where
c is the number of clusters per arm,
y is the number of person-years per cluster,
λ0 is the rate in the control group,
λ1 is the rate in the treatment group,
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k is the coefficient of variation in both groups,
α is the alpha level,
β and is the type II error rate.
• The base rate for mortality is assumed to be 0.02 per year in the target age range.
• We assume the same coefficient of variation for village-level mortality as for the Niger
site, i.e. 0.34.
• We assume that 16.7% of the population is in the target range of 1–59 months.
• We assume an average community size of 1000 people.
• We assume 10% loss to follow-up per year.
• The effect size is 0.1365.
The number of clusters per arm for Burkina Faso is 170, and the total number of clusters
for Burkina Faso is 340.
The sample size is fixed at 341 clusters at the begining of the study.
We estimate that 341 clusters will provide 80% power to detect a 13.65% reduction in
mortality in 1-59 month age group.
Power for secondary outcomes
All analyses assume a two-sided alpha of 0.05 and are anticipated to provide a power of at
least 80%.
nutritional status The proposed sample size will be sufficient to detect a standardized
effect size of 0.108 for weight and height (in the HDSS communities), and of 0.0401 for
MUAC (in all communities). We assume an ICC of 0.015.
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malaria We assume a placebo parasitemia of 0.2 and an ICC of 0.056. We anticipate at
least 80% power to detect a difference of 0.083 between the arms.
antibiotic resistance We assume a placebo drug resistance of 0.05 and an ICC of 0.1. We
anticipate at least 80% power to detect a difference of 0.082 between the arms.
4
Randomization
Of the 341 communities available in Nouna district, 48 of these fall within an existing
HDSS. We will stratify the randomization within this group.
Considering only the 48 HDSS communities, note that an unstratified randomization
which allocated each community to Azithro with probability 1/2 would yield an approxi-
mate 3:2 or worse imbalance (in either direction) with a probability of approximately 8.2%.
The randomization will be conducted using R. The function sample with option replace=FALSE
will be used to conduct the random shuffle. Note that the choice of the random number seed
completely determines the randomization. To ensure the integrity of the randomization, we
will use the procedure we used for MORDOR/Malawi.
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5
Abbreviations
ANCOVA Analysis of covariance
DSMC Data and Safety Monitoring Committee
HAZ height for age Z score
HDSS Health and demographic surveillance system
MUAC Mid upper arm circumference
SAP Statistical Analysis Plan
WAZ weight for age Z score
WHZ weight for height Z score
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6
Revision History
5 Oct 2018 Another two pre-specified secondary analyses were added: 1. the number of
azithromycin doses received; 2. factorial analysis.
1 Aug 2019 Power calculation and sample size updated.
16 Jan 2020 Changed the title to CHAT.
28 Jan 2020 Took the sensitivity analysis out from Statistical considerations under 2.1.1
Mortality Trial at page 5.
15 June 2022 Changed person time calculation to assign 0 person-time to individuals who
are lost to followup or missing during the inter-census interval.
13 Feb 2023 Specified interaction on the additive scale; specified the by-phase subgroup
analysis.
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