Clinical trial NCT03338244
Conditions: Childhood Mortality
Keywords: Childhood mortality Mass treatment Azithromycin Infection
Sponsor: University of California, San Francisco
Start date: 2017-10-20; Completion date: 2020-07-31
Enrollment: 66228 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
MORDOR was a cluster-randomized placebo controlled trial that assessed the efficacy of mass azithromycin distributions for the prevention of childhood mortality. All communities were subsequently treated with mass azithromycin for one year. The present trial re-randomized communities to azithromycin or placebo for the fourth and fifth year of the study.

Detailed description:
Pre-trial treatments: in the original MORDOR trial, communities were randomized to 4 rounds of biannual mass distributions of either azithromycin or placebo (i.e, the first two years of the trial). All communities were subsequently treated with 2 rounds of biannual mass azithromycin (i.e., the fifth and sixth rounds, during the third year of the trial).

Present trial design: The present trial enrolls all communities previously enrolled in the Niger site of MORDOR, and re-randomizes communities to biannual mass distributions of either azithromycin or placebo (i.e., the seventh, eighth, ninth and tenth rounds of mass drug administration since randomization for the original MORDOR trial, occurring in the fourth and fifth year since randomization). This results in four patterns of community-based treatment: four years of azithromycin (AAAAA), three years of azithromycin followed by one year of placebo (AAAPP), two years of placebo followed by two years of azithromycin (PPAAA), and two years of placebo followed by one year of azithromycin and then one year of placebo (PPAPP). The primary outcome will be all-cause mortality in children aged 1-59 months,as determined by biannual census. The mortality rate following re-randomization (i.e., the seventh, eighth, ninth and tenth rounds of mass drug administration) will be compared between the two re-randomized groups.

Arms: arm 1: Communities will receive four rounds of biannual mass azithromycin. arm 2: Communities will receive four rounds of biannual mass placebo.
Interventions: intervention 1: Azithromycin intervention 2: Placebo
Intervention descriptions: intervention 1: Children aged 1 month to 60 months per community will be offered weight or height-based, directly observed, oral azithromycin suspension every 6 months intervention 2: Children aged 1 month to 60 months per community will be offered weight or height-based, directly observed, oral placebo suspension every 6 months

Protocol documents:
Prot_000.pdf: 
version 1.10 
 
MORDOR Study 
Manual of Operations and Procedures 
 
Dr. Tom Lietman 
1 | P a g e  
 
 
 
Mortality Reduction After 
Oral Azithromycin 
 
Manual of Operations and Procedures 
 
The Francis I. Proctor Foundation, Global Health Sciences 
University of California, San Francisco 
The Carter Center 
Niger Ministry of Health 
 
The Dana Center, Bloomberg School of Public Health 
Johns Hopkins University 
Tanzania National Institute for Medical Research and Muhimbili Medical 
Center 
 
The London School of Hygiene & Tropical Medicine 
College of Medicine, University of Malawi, Blantyre 
Malawi Ministry of Health 
 
NCT NUMBER 03338244 
Date of document Oct 1st 2019 


 
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Table of Contents 
ABBREVIATIONS ..................................................................................................................................... 5 
CHAPTER 1: OVERVIEW .......................................................................................................................... 6 
1.1. 
EXECUTIVE SUMMARY ....................................................................................................................... 6 
1.2. 
OBJECTIVES ..................................................................................................................................... 6 
1.3. 
STUDY SITES .................................................................................................................................... 7 
CHAPTER 2: CONTEXT .................................................................................................................... 7 
CHAPTER 3: STUDY DESIGN ....................................................................................................... 10 
3.1. 
RANDOMIZATION ........................................................................................................................... 11 
CHAPTER 4: STUDY ELIGIBILITY ................................................................................................ 11 
4.1. 
ELIGIBLE COMMUNITIES ................................................................................................................... 11 
4.2. 
ELIGIBLE INDIVIDUALS ..................................................................................................................... 12 
4.3. 
STUDY SCHEDULE ........................................................................................................................... 13 
CHAPTER 5: CORE AND NON-CORE STUDY ELEMENTS ...................................................... 14 
5.1. 
MORTALITY STUDY ......................................................................................................................... 14 
5.1.1. 
Core Elements (Malawi, Niger, and Tanzania) ............................................................. 14 
5.2. 
MORBIDITY/RESISTANCE STUDY ........................................................................................................ 15 
5.2.1. 
Core Elements (Malawi, Niger, and Tanzania) ............................................................. 15 
5.2.2. 
Non-core Elements ............................................................................................................ 15 
CHAPTER 6:  CENSUS ..................................................................................................................... 16 
6.1. 
CENSUS ........................................................................................................................................ 16 
6.2. 
RANDOM CENSUS VERIFICATION ....................................................................................................... 18 
6.3. 
VERBAL AUTOPSY .................................................................................................................... 19 
6.4. 
VALIDATING THE MORTALITY OUTCOME .............................................................................. 20 
6.4.1 Niger .......................................................................................................................................... 20 
6.4.2 Malawi ..................................................................................................................................... 21 
6.4.3 Tanzania .................................................................................................................................. 21 
CHAPTER 7: REGISTERING PARTICIPANTS FOR SPECIMEN COLLECTION .................... 22 
CHAPTER 8: BLOOD SAMPLES .................................................................................................... 22 
8.1. 
FINGERSTICK ............................................................................................................................ 23 
8.2. 
DRIED BLOOD SPOT FOR MALARIA ....................................................................................... 24 
8.3. 
HEMOGLOBIN TEST ................................................................................................................. 26 
8.4. 
THICK AND THIN SMEARS FOR MALARIA ............................................................................. 27 
8.5. 
MATERIALS FOR BLOOD COLLECTION ................................................................................................. 28 
CHAPTER 9: SPECIMEN COLLECTION FOR RESISTANCE TESTING .................................. 29 
9.1. 
POPULATION ................................................................................................................................. 29 
9.2 NASOPHARYNGEAL SWABS ....................................................................................................................... 29 
9.2.1 
Materials for Swab Collection for Resistance Testing ................................................ 30 
9.2.2 
Protocol for Tubing and Handling of Samples .............................................................. 31 
9.3 
STOOL SAMPLES ................................................................................................................................. 31 
9.3.1 
Stool Specimen Collection ................................................................................................ 32 
9.3.2 
Materials for Stool Specimen Collection ....................................................................... 33 


 
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9.4 
QUALITY CONTROL MEASURES FOR SPECIMEN COLLECTION ........................................................................ 34 
CHAPTER 10: COST-EFFECTIVENESS ......................................................................................... 35 
10.1. 
COSTS .......................................................................................................................................... 35 
10.2 
EFFECTIVENESS .............................................................................................................................. 35 
CHAPTER 11: TRAINING ............................................................................................................... 36 
11.1 
STANDARDIZATION ......................................................................................................................... 36 
11.2 
PILOT STUDIES ............................................................................................................................... 36 
CHAPTER 12: SAMPLE ORGANIZATION, TRANSPORT, AND STORAGE .......................... 36 
12.1 
DE-IDENTIFICATION ........................................................................................................................ 37 
12.2 
SPECIMEN TRANSPORT ........................................................................................................... 37 
12.3 
SPECIMEN STORAGE ................................................................................................................ 38 
12.3.1 
Short-term Sample Storage ......................................................................................... 38 
12.3.2 
Long-term Sample Storage .......................................................................................... 38 
12.4 
CATALOG SPECIMENS ............................................................................................................. 38 
CHAPTER 13: MICROBIOLOGY AND MOLECULAR LABORATORY PROCEDURES ........ 38 
13.1 
STORAGE AND TRANSPORT MEDIA ....................................................................................... 38 
13.1.1 
Preparation of STGG Transport Medium ................................................................. 38 
13.1.2 
Preparation of Amies Transport Medium ................................................................. 39 
13.1.3 
Preparation of Nutrient Agar Medium/Slants ......................................................... 41 
13.2 
METHODS FOR MALARIA TESTING ........................................................................................ 41 
13.2.1 
Blood Smears ................................................................................................................. 41 
13.2.2 
Dried Blood Spots ......................................................................................................... 42 
13.3 
METHODS FOR RESISTANCE TESTING ................................................................................... 48 
13.3.1 
Isolation & Identification of Organisms Using Microbiological Techniques ..... 48 
13.3.2 
Microbiological Resistance Testing for Swabs ........................................................ 50 
13.3.3 
Nucleic Acid Based Resistance Testing for Stool Specimens .................................. 52 
13.4 
METHODS FOR CHLAMYDIA TRACHOMATIS (NON-CORE) ...................................................... 54 
13.4.1 
Description of Abbott RealTime Assay ..................................................................... 54 
13.4.2 
Processing of Samples for PCR ................................................................................... 54 
13.4.3 
Procedure for Masking Samples ................................................................................. 54 
13.4.4 
Procedure for Pooling Samples ................................................................................... 55 
13.4.5 
Quality Control ............................................................................................................ 55 
13.4.6 
Quantification .............................................................................................................. 55 
CHAPTER 14: STUDY MEDICATION ........................................................................................... 56 
14.1 STUDY MEDICATION DESCRIPTION (FROM PFIZER, INC.) ............................................................................... 56 
14.1 
DOSAGE INFORMATION ................................................................................................................... 56 
14.2 
MEDICATION PROCUREMENT/DONATION ........................................................................................... 56 
14.3 
MEDICATION QUALITY CONTROL ....................................................................................................... 57 
14.4 
ANTIBIOTIC DISTRIBUTION & MONITORING COVERAGE ......................................................................... 57 
14.5 
ADVERSE REACTIONS/SIDE EFFECTS ................................................................................................... 57 
14.6 
ADVERSE EVENTS SYSTEMS .............................................................................................................. 58 
14.6.1 
Passive Adverse Events Monitoring .......................................................................... 58 
14.6.2 
Active Adverse Events Monitoring ............................................................................ 58 
14.6.3 
Adverse Events Data .................................................................................................... 60 
14.8  SUPPLY ISSUES ..................................................................................................................................... 60 
CHAPTER 15: PROTECTION OF HUMAN SUBJECTS ............................................................... 61 


 
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15.1 
INSTITUTIONAL REVIEW BOARD APPROVAL .......................................................................................... 61 
15.1.1. 
Niger ............................................................................................................................... 61 
15.1.2 
Tanzania ........................................................................................................................ 62 
15.1.3 
Malawi ........................................................................................................................... 62 
15.2 
INFORMED CONSENT ...................................................................................................................... 62 
15.3 
RISKS AND BENEFITS OF STUDY PROCEDURES ....................................................................................... 63 
15.3.1 
Verbal Autopsy .............................................................................................................. 63 
15.3.2 
Swabbing Procedures ................................................................................................... 63 
15.3.3 
Stool Collection ............................................................................................................ 63 
15.3.4 
Blood Testing ................................................................................................................. 63 
15.3.5 
Anthropometric Measurements .................................................................................. 63 
CHAPTER 16: STUDY MONITORING .......................................................................................... 64 
CHAPTER 17: DATA AND SAFETY MONITORING COMMITTEE CHARTER ..................... 64 
17.1 
PRIMARY RESPONSIBILITIES OF THE DSMC .......................................................................................... 64 
17.2 
DSMC MEMBERSHIP .............................................................................................................. 65 
17.3 
CONFLICTS OF INTEREST ......................................................................................................... 65 
17.4 
TIMING AND PURPOSE OF THE DSMC MEETINGS ............................................................... 65 
17.5 
PROCEDURES TO ENSURE CONFIDENTIALITY AND PROPER COMMUNICATION .............................................. 66 
17.6 
STATISTICAL MONITORING GUIDELINES .............................................................................................. 67 
17.7 
DSMC CONTACT INFORMATION ....................................................................................................... 68 
CHAPTER 18: DATA COLLECTION, MANAGEMENT, AND SECURITY .............................. 69 
18.1. 
SCOPE OF DATA ............................................................................................................................. 69 
18.2 
DATA STORAGE, MANAGEMENT, AND SECURITY ........................................................................ 70 
REFERENCES .................................................................................................................................... 71 
APPENDIX ........................................................................................................................................ 75 
 


 
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Abbreviations 
 
DCC: Data Coordinating Center  
GPS: global positioning system 
IRB: Institutional Review Board 
JHU: The Johns Hopkins University  
LSHTM: London School of Hygiene and Tropical Medicine 
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 


 
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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 Recently, we demonstrated that mass azithromycin 
distributions for trachoma may have the unintended benefit of reducing 
childhood mortality.2,3 This surprising result was observed in an area of Ethiopia 
with highly prevalent trachoma. Another trial is necessary to determine whether 
a similar effect will be seen in areas not eligible for trachoma treatments. If mass 
azithromycin can be proven to reduce childhood mortality, then widespread 
implementation in developing countries could be feasible and perhaps integrated 
with other interventions.  
 
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 multi-site, cluster-randomized trial comparing communities 
randomized to oral azithromycin with those randomized to placebo. To assess 
the generalizability of the intervention, we will monitor for antibiotic resistance, 
which could potentially limit adoption of mass antibiotic treatments. We will 
also begin to investigate possible explanatory factors for mortality reduction by 
assessing several measures of the burden of infectious diseases. We hypothesize 
that mass azithromycin treatments will reduce childhood mortality even in areas 
with low trachoma prevalence, and will be accompanied by an acceptable level 
of antibiotic resistance.  
 
1.2. 
Objectives  
 
1: To test the hypothesis that mass distributions of oral azithromycin 
targeted to 1-60 month old children reduces childhood mortality, in a 
cluster-randomized trial 
 
2: To assess the cost-effectiveness of mass azithromycin in reducing 
childhood mortality  
 
3: To investigate the most plausible factors that could explain an effect of 
mass azithromycin on childhood mortality  
 
4: To assess for emergent macrolide resistance among children following 
mass azithromycin treatments 
5: To collect and securely store samples from the nasopharynx, nares, 
conjunctiva, blood, and stool after azithromycin distributions to assess 
infectious burden and resistance 


 
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Ancillary Aim 1 (non-core): To assess the effect of mass azithromycin 
treatments on microbial diversity in the conjunctival, nasopharyngeal, 
nasal, and intestinal biome of children 
 
1.3. 
Study Sites 
A consortium of experienced researchers and program implementers will 
conduct the study in 3 sites (Malawi, Niger, and Tanzania), chosen for diversity 
of geography and comorbidities, existence of a functioning national azithromycin 
distribution program, and previous investigator experience: 
• Southern Africa: Mangochi district, Malawi (Malawi Ministry of Health, 
University of Malawi, Blantyre, and London School of Hygiene & Tropical 
Medicine) 
• West Africa: Loga and Boboye departments, Niger (Carter Center Niger, 
Niger Ministry of Health, University of California, San Francisco) 
• East Africa: Kilosa and Gairo districts, Tanzania (National Institute for 
Medical Research, Kilosa, Johns Hopkins University) 
Chapter 2: Context 
Reduction of childhood mortality is an international development goal. The 
United Nations Millennium Development Goals call for a two-thirds reduction in 
childhood mortality by 2015.4 Despite progress this past decade, under-5 
mortality remains unacceptably high.1,5,6 In Africa, where the burden of 
childhood mortality is greatest, three-quarters of all childhood deaths occur after 
the neonatal period, with malaria, pneumonia, and diarrhea accounting for the 
majority.7 Management is often case detection and individualized therapy, but 
novel strategies such as mass treatment may play a role.8-10  
 
The proposed research will assess whether childhood mortality can be reduced 
using a novel approach—mass antibiotic administrations similar to those used 
for trachoma. The World Health Organization (WHO) recommends repeated 
annual distribution of single-dose oral azithromycin to each resident of 
trachoma-endemic communities, though more frequent treatments may be 
necessary.11-13  While azithromycin effectively clears the ocular strains of 
chlamydia that cause trachoma,12,14,15 the drug also has activity against major 
causes of childhood mortality, including respiratory infections, diarrhea, and 
malaria.16-18 Mass treatments have been shown to be effective for these non-
chlamydial infections. For example, in a  study we conducted in Nepal, the 
prevalence of diarrhea in children decreased from 32% before a mass 
azithromycin treatment, to 11% ten days post-treatment (OR 0.26 [95% CI 0.14 – 
0.43]).19 Others in our consortium have found that malaria parasitemia decreased 
by 44% after mass azithromycin treatments.20 It is feasible that periodic mass 


 
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azithromycin distributions could reduce the burden of these infections 
sufficiently enough to reduce childhood mortality.2  
 
Mass azithromycin treatments targeted to children may maximize the benefit 
of antibiotics, while limiting resistance. Pre-school children have the most to 
gain from mass azithromycin treatment because the burden of treatable, life-
threatening infection is highest in this age group.21 Treatment of children may 
also provide indirect protection to untreated members of the community due to a 
herd effect.22 In a trial in Ethiopia, quarterly mass azithromycin treatments 
targeted to children aged 1-10 years resulted in a 47% reduction in ocular 
chlamydia in untreated community members.23 Although we demonstrated this 
indirect protection for ocular chlamydial infection, other infectious diseases may 
be similar. Reducing the infectious burden of respiratory pathogens, malaria, and 
diarrhea might benefit children who missed a distribution or were too old for 
treatment. Given that community antibiotic resistance correlates with the volume 
of antibiotic consumption,24-26 targeting children may result in less antibiotic 
resistance in a community. Targeting children would also be easier logistically 
and require fewer doses of donated antibiotic, both of which lower costs of 
administrations.  
 
The finding of a reduction in childhood mortality after mass azithromycin 
treatment warrants replication in other settings to increase generalizability. 
The current proposal differs from our earlier trial in several important aspects. 
First, we will conduct the study in areas with little or no trachoma to enhance 
generalizability. Second, we will monitor a subset of communities for measures 
of infectious and nutritional morbidity, to identify explanatory factors for any 
mortality reduction. Third, we will randomize control communities to placebo, 
reducing the chance of differential co-intervention and differential outcome 
assessment. Fourth, we will treat children 1 month and older, as opposed to 12 
months and older. Fifth, we will test whether restricting azithromycin to only 
children will limit selection of community antibiotic resistance in Streptococcus 
pneumoniae and other organisms. Sixth, we will monitor for a longer period of 
time than the previous study, to assess whether mass azithromycin results in a 
more sustained mortality reduction. Finally, we will enroll a larger number of 
children and randomize a larger number of clusters, providing considerably 
more statistical power to detect even a smaller reduction in mortality. 
 
Mass drug administrations can be implemented relatively easily and 
potentially have a major impact on morbidity and mortality. Mass treatments 
are simple interventions and considerable expertise in their deployment already 
exists in developing countries.27 They can have a dramatic impact in some 
settings. For example, a study of mass distribution of vitamin A for 
xerophthalmia in the 1980s found that treated communities had lower childhood 


 
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mortality compared to untreated communities.28 Trials in the following decade 
largely confirmed these results.29-34 Now, vitamin A distributions are provided in 
most countries that have high childhood mortality, with a majority of programs 
achieving the targeted 70% coverage.35 A report estimated that vitamin A could 
prevent as many as a quarter million childhood deaths each year.36 This same 
study estimated that treatment of diarrhea, sepsis, pneumonia, and malaria 
could prevent over 3 million deaths each year. If mass azithromycin treatments 
can prevent or treat even a fraction of these infections, this could have a 
tremendous effect on childhood mortality.    
 
Millions of azithromycin treatments are currently distributed each year. Since 
1999, over 225 million treatments of azithromycin have been donated through 
the International Trachoma Initiative for distribution by local partners in 19 
countries.37 Many Ministries of Health and non-governmental organizations 
have experience procuring and transporting the drug, organizing local health 
workers for antibiotic distribution, and monitoring antibiotic coverage. These 
activities have proven to be sustainable; excluding the cost of the antibiotic itself, 
mass distribution costs an estimated $0.25-0.40 per person treated.38 Although 
the costs of azithromycin would add to this, generic azithromycin is available, 
and economies of scale would likely reduce drug costs if mass treatments became 
more widespread. 
 
 
 


 
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Chapter 3: Study Design 
We will assess childhood mortality over two years, comparing communities 
where children aged 1-60 months receive biannual oral azithromycin (“Azithro” 
arm), to communities where the children receive biannual oral placebo 
(“Control” arm). We will also randomize a separate set of communities to these 
treatment strategies, but monitor instead for infectious disease morbidity and 
macrolide resistance (“Azithro-Plus" and "Control-Plus”). These communities 
will not be included in the mortality comparison, since identification and 
treatment of morbidities could interfere with any mortality effect of 
azithromycin. 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. 
 
Figure 1: Trial Profile 
 
  
 
 
N + 30 communities 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
  
MORTALITY 
N communities 
 MORBIDITY/RESISTANCE 
30 communities 
 
  
 
 
 
 
 
 
 
 
 
 
 
  
Azithro 
 
Control      Azithro-Plus 
 
Control-Plus  
TREATMENT 
 
 
Biannual  
mass oral 
azithromycin 
to children 
 
 
Biannual 
mass 
oral placebo 
to children 
 
Biannual  
mass oral 
azithromycin to 
children 
 
Biannual mass 
oral placebo to 
children  
 
N/2   
communities 
 
N/2 
communities 
15 
communities 
 
15  
Communities 
 
 
  
 
 
 
  
 
 
 
 
MONITORING 
 
 Every 6 months: 
• 
Census (births, 
deaths, pregnancies, 
migration) 
• 
Verbal Autopsy 
(cause of death) 
• 
Costs of census and 
treatments 
 Every 12 months: 
• 
Blood smears, blood spots, 
and hemoglobin 
• 
Nasopharyngeal swabs 
• 
Nares swabs  
• 
Stool samples or rectal 
swabs 
• 
Conjunctival swabs  
• 
Anthropometry 
 


 
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3.1. 
Randomization  
 
Randomization of Treatment Allocation. In each site, communities within a 
contiguous area of 300,000 to 600,000 individuals will be randomized to 
azithromycin or placebo for the mortality study. An additional 30 communities 
will be randomized into one of two study arms for embedded, cost-efficient, 
morbidity studies. Since mortality and morbidity study communities will be 
drawn from the same pool, findings from one study will be relevant to the other. 
Each site has a different set of prevalent co-morbidities and different access to 
health interventions. 
 
No stratification or block randomization is planned within each country. The 
allocation of community assignments will be conducted for each country 
separately using simple random sampling. For each country, all study arms 
(mortality and morbidity) will be assigned from the same list of eligible 
communities. A pseudorandom number scheme will be implemented using the 
statistics package R. Randomization will be conducted by T. Porco. Procedural 
and algorithmic details are provided in an appendix to the Statistical Analysis 
Plan. 
 
Study Participants: At months 0, 12, and 24, 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.  
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 of between 200–2,000 
people. 
4. The community is not in an urban area.  


 
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4.2. 
Eligible Individuals 
 
Mortality Study:  
Census: The study can be thought of as consisting of four 6-month 
segments, each of which starts with a census and ends with a follow-up 
census. All children in the study communities aged 1-60 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 ≥ 60months) 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.   
 
Morbidity/Resistance Study:  
 
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-
60 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. 
 
 
 
 
Note: All individuals must be registered on the official  
census in order to be considered part of the study. 


 
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4.3. 
Study Schedule 
The schedule for examination and treatment is shown below in Table 1:  
MORDOR Study Schedule 
 
  
MORTALITY 
MORBIDITY / RESISTANCE 
Azithro 
Control 
Azithro-Plus 
Control-Plus 
Oral 
Azithromycin  
(N/2 
communities) 
Oral Placebo        
(N/2 
communities) 
Oral Azithromycin 
(N = 15 communities) 
Oral Placebo    
(N = 15 communities) 
MORDOR 0 
Jan-15 to Jul-
15* 
Census 
Azithro 
Census 
Placebo 
Census 
Swabs  
Blood 
Stool 
Anthropometry  
Azithro 
Census 
Swabs  
Blood 
Stool 
Anthropometry  
Placebo 
MORDOR 6 
Aug-15 to 
Jan-16 
 
Census 
Verbal Autopsy 
Azithro 
Census 
Verbal Autopsy 
Placebo 
Census 
Azithro 
NP Swabs, Blood, Stool a 
Malaria Collectionb 
Census 
Placebo 
NP Swabs, Blood, Stool a 
Malaria Collectionb 
MORDOR 12 
Feb-16 to Jul-
16 
Census 
Verbal Autopsy 
Azithro 
Census 
Verbal Autopsy 
Placebo 
Census 
Swabs  
Blood 
Stool 
Anthropometry  
Azithro 
Census 
Swabs  
Blood 
Stool 
Anthropometry  
Placebo 
MORDOR 18 
Aug-16 to 
Jan-17 
Census 
Verbal Autopsy 
Azithro 
Census 
Verbal Autopsy 
Placebo 
Census 
Azithro 
NP Swabs, Blood, Stool a 
Census 
Placebo 
NP Swabs, Blood, Stool a 
MORDOR 24 
Feb-17 to Jul-
17 
Census 
Verbal Autopsy 
Azithroc 
Census 
Verbal Autopsy 
Azithroc 
Census 
Swabs 
Blood 
Stool 
Anthropometry 
Azithroc 
Census 
Swabs  
Blood 
Stool 
Anthropometry 
Azithroc 
Only children aged 1 month to 60 months per community will be treated 
Arms Azithro+ & Control+: swabs, blood testing, and stool samples or rectal swabs from 40 randomly selected children aged
60 months per community; anthropometry from a cohort of children aged 1 to 60 months per community; NP swabs from 40 
individuals aged 7 – 12 years (Niger only) 
 
a: Tanzania only 
b: Niger only  
c: Decision to treat ALL communities with azithromycin at 24-months if there is a significant mortality difference (in 
preparation for possible contingent study, Niger only) 
*: +/- 1 month 


version 1.10 
 
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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 
 
5.1.1. Core Elements (Malawi, Niger, and Tanzania) 
In all three study sites, we will conduct the following study activities for the 
mortality study: 
Training 
Before the baseline visit, the University of California San Francisco (UCSF) team will 
certify local team members at all three study sites. Standardization activities before 
each biannual census will consist of didactic classroom instruction and mock census 
activities, followed by in-field training to harmonize protocols.  
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 
women will be noted at each census, to maximize inclusion of newborns on the 
subsequent census. 
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 team identified all births, deaths, and 
migratory episodes relative to the previous census. 
Verbal Autopsy 
Each of the study sites will perform a verbal autopsy for all children (aged 1-60 
months) identified as having died during the study.39  
Treatment 
Children aged 1-60 months on the current census will be offered weight- or height-
based (<1 year weight and children who cannot stand; ≥1 year height), directly 
observed, oral azithromycin suspension (or oral placebo) every 6 months for 2 years 
as performed in trachoma programs in each of the 3 countries. Specifically, 
individuals are eligible on or after their one month birthday, and prior to the day of 
their fifth birthday.  


 
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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, and 18, we will 
identify any children who have missed 2 or more consecutive treatments, and 
forward this information to the census team. 
5.2. 
Morbidity/Resistance Study 
 
5.2.1. Core Elements (Malawi, Niger, and Tanzania) 
 
All core elements described in 5.1.1 will also be conducted in the morbidity / 
resistance study.  The enumerated population census for the 
morbidity/resistance communities will include 0-12 year olds.   
 
The designation “core elements” means that all study sites will be performing 
the study activity.  
 
In all study sites, we will perform the following tests on a random sample of 40 
children aged 1-60 months from each Azithro-Plus and Control-Plus community 
at baseline, 12, and 24 months:  
- 
Blood samples (dried blood spots optional) for malaria and anemia 
- 
Nasopharyngeal swabs for pneumococcal macrolide resistance 
- 
Stool samples or rectal swabs to assess for macrolide resistance 
- 
Assessment of costs of a census and mass drug administration (at 6 or 12 
and 24 month visits only) 
 
Samples will be processed in-country when possible; otherwise they will be 
shipped to UCSF. 
 
5.2.2. Non-core Elements 
The designation “non-core” element means that study sites may choose if they 
wish to perform the study activity. If the study sites perform the study activity, 
the methodology will be performed according to this protocol so that the 3 sites 
may be more easily compared. Sites may choose to perform several non-core 
morbidity assessments at the 0, 12, and 24 month visits: 
- 
Nasal swabs for macrolide resistance in Staphylococcus aureus 
- 
Conjunctival swabs for Chlamydia trachomatis 
- 
Conjunctival examination and photography to assess for trachoma 
- 
Anthropometric assessments 
- 
Nasopharyngeal swab collection at local health posts to compare 
macrolide resistance in the community with that in the health post 
 


 
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In addition, there are several non-core activities that occur outside the annual 
morbidity/resistance assessment: 
- 
Additional study visit during the first malaria season to collect blood 
samples for malaria and anemia testing 
- 
Clinic-based case finding for childhood illnesses at all health clinics in the 
study area 
 
Costs 
We anticipate recording the cost of distributions in a random subset of 12 villages at 
the 6 or 12 and 24 month visits. 
Details of the non-core elements for each study site can be found in the 
Appendices. 
 
Chapter 6:  Census  
6.1. 
Census 
Census Team 
Census workers will be selected by the site study coordinator. These individuals 
may have different qualifications and educational backgrounds in the 3 sites, 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. Census 
workers will ideally be local community dwellers, and familiar with the 
communities being enumerated. 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, 
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 (or 0-12 in morbidity 
communities) 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 


 
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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 
  
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 local SIM card with data plan can be purchased for each tablet, 
and the data uploaded via cell towers twice per day (midday and 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, 


 
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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 site 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 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 
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 5 study 
visits per country (approximately 40 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 


 
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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).   
 
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 
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 trial biostatistician 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 
in turn 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. 
 


 
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Questionnaire Administration 
Each site will choose between the full-length WHO questionnaire (2007 version) 
or an abbreviated form (which utilizes questions from both WHO 2007 and 2012), 
translated into the local language as appropriate. 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 2 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.  
 
Translation 
The verbal autopsy will be translated into the local language by a professional 
translator and then back-translated into English by a second translator. 
Discrepancies that are noted in the back-translation will be provided to the 
original translator, and adjustments made as needed.  
 
At each country site, the questionnaire will be validated and reviewed to ensure 
that it is culturally appropriate.  
 
6.4. 
Validating the mortality outcome 
 
6.4.1 Niger 
Deaths from the biannual census will be validated from local death registries. In 
an effort to improve the quality of existing death registries in villages and health 
facilities, the Study Coordinator will conduct sensitization activities in study 
communities and CSIs before the study begins. During sensitization, the Study 
Coordinator will work closely with local village chiefs, mayors, and health 
centers to develop a strategy for obtaining more accurate death registers during 
the study.  
 
CSIs and villages maintain local death registries. Health professionals record 
deaths that occur in the CSI. The CSI death registries include: name, age, village, 


 
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cause of death. The village chief also maintains a death registry in the village. 
The village death registry includes: name, age, village, cause of death. 
 
The Study Coordinator will visit each CSI and take photos of local death 
registries with all deaths from children aged 0-9 years. We collect a larger age 
group than that collected in the census since age misclassification may occur 
either on the census or on the death registry. The Study Coordinator will keep all 
information present in the registry for all children aged 0-9 years, including 
name and village.  
 
The Data Manager will be responsible for linking the death registry file with the 
census file, using the name, age, and village of deceased children. We expect that 
both the census and the registry will miss deaths.  
 
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 death registry. Note that the 
deaths from the death registry will not be used in the primary outcome.  
 
6.4.2 Malawi 
Deaths will be identified in 2 ways: from the biannual census, and from the key 
informant system. The trial biostatistician 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; the 
community name; and the father’s and mother’s names. The study coordinator 
will in turn 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 child will be located in the census 
database. We will provide phone credit as appropriate for the key informants. 
 
Trained verbal autopsy interviewers will be responsible for a distinct geographic 
area, and will be responsible for being in regular contact with the key informant 
from each community. We will aim to perform all verbal autopsies within 2 
months of death. The interviewee 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 the home 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 Chichewa, Yao or English, as appropriate, on a 
tablet computer. 
 
6.4.3 Tanzania 


 
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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.   
Community Monitors will visit a random sample of households identified on the 
census as having children. They will visit their assigned households each month 
to monitor for deaths. These records will be kept apart from the data collected by 
the census teams (at six month intervals). The two lists will be compared and any 
discrepancies followed up.     
 
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:  
1) Thick and thin smears, assessed for malaria parasitemia and 
gametocytemia by trained local 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) and sent to the CDC for serological testing.  
 


 
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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!  
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 
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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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 Malaria 
 
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. 
 
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.  
 
 
8.5. 
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 


 
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Manual of Operations and Procedures 
 
Dr. Tom Lietman 
29 | P a g e  
 
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 
 
Google Nexus 7 
External battery pack 
Chapter 9: Specimen Collection for Resistance Testing  
 
9.1. 
 Population 
We will collect nasopharyngeal and stool samples on a random set of 40 children 
aged 1-60 months from each of the communities in the Morbidity study. 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. 
 
 
9.2 Nasopharyngeal Swabs 
Nasopharyngeal swabs will be stored in DNA/RNA shield media by Zymo or 
STGG media, and standard microbiologic techniques will be used to isolate S. 
pneumoniae and test for resistance to azithromycin, penicillin, and clindamycin. 


 
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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 
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). Nasal sites 
will be swabbed with a sterile, Dacron polyester-tipped swab with a plastic shaft 
(manufactured by Fisherbrand).   
 
Sample Tubes 
All field samples for DNA testing will be collected into sterile 2.0ml 
microcentrifuge tubes, manufactured by Sarstedt®. (DNA-free tubes will be used 
for collection in DNA/RNA shield.) 
 
 
Cooler Bags with Frozen Ice Packs 


 
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Dr. Tom Lietman 
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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 local health center 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 Health Center, which is under 24-hour security 
guard supervision. 
 
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 local 
microbiology laboratory and/or to the US for processing.  The person in charge 
of labeling, tubing, arranging, and handling the samples needs to perform this 
task in the most orderly and attentive manner. 
 
1. Both hands of the tuber should be gloved at all times. The tuber's gloves 
only need to be changed when any potential contamination of the gloves 
occurs. The tuber opens the capped, hinged lid of a microcentrifuge tube, 
which has been labeled with the participant’s random identification 
number. 
2. The swab is inserted by the examiner into the microcentrifuge tube held 
by the tuber.  The swab shaft should only be inserted until the swab head 
is fully in the tube. The tuber will cut the swab shaft with sterile scissors.  
3. The tuber should screw the cap of the microcentrifuge tube tightly, flick 
the tube to mix the sample with the media (for tubes with DNA/RNA 
shield media), and place it in the sample collection box, located in the 
cooler bag filled with frozen ice packs. The flap of the cooler bag should 
be closed between each patient.  The cooler bag should be in as cool a 
place as possible in the field, in a shaded area out of the sun. 
Upon returning from the field each day, the samples in STGG will be 
immediately taken to a local health center and stored in a commercial -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 


 
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Manual of Operations and Procedures 
 
Dr. Tom Lietman 
32 | P a g e  
 
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.43  
 
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.45  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,46-47 or exploring the complete microbiome of the 
stool specimens. 
 
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 


 
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Dr. Tom Lietman 
33 | P a g e  
 
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. 
 
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 
 


 
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Manual of Operations and Procedures 
 
Dr. Tom Lietman 
34 | P a g e  
 
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 
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. 
 
 
9.4 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.  
 


 
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Dr. Tom Lietman 
35 | P a g e  
 
 
Chapter 10: Cost-Effectiveness  
 
10.1. 
Costs 
We will record the costs of distributions in a random subset of 12 villages at 
MORDOR 6 or 12 and MORDOR 24. We will assume a societal perspective. 
Programmatic costs will be included, such as: azithromycin, antibiotic 
distributors, supervisors, drivers, fuel, and the time of community volunteers, 
and the costs of azithromycin will be estimated using the market price for 
generic formulations. See appendix for the study forms to be used. Collection of 
certain costs will be incorporated into the electronic data capture system, 
including the duration of time that the antibiotic distributors worked, the 
number of antibiotic distributors per team, the number of doses of 
azithromycin/placebo given, and the number of doses of azithromycin/placebo 
wasted. Other costs will be collected on paper forms from the appropriate 
parties: the drivers will complete paper forms describing costs of fuel and 
automobile maintenance, and the study coordinator will complete paper forms 
describing the costs of consumables purchased for the study, as well as the 
overhead costs of maintaining the mass drug administration office. 
 
10.2 
Effectiveness 
To estimate effectiveness, we will estimate the childhood mortality rate 
differently from the primary outcome of the study, in that we will account for all 
person-time in the community for children aged 1-60 months. Children who are 
born or move into the study area after the initial census will have their person-
time counted in the mortality outcome, regardless of whether they are 
enumerated at the previous census. Similarly, we will count all childhood deaths 
in the study area, even if the child never appears on the census. The effectiveness 
outcome will therefore include children who are born and die within an inter-
census interval. Estimating the effectiveness will require several aspects from the 
census data collection: 
• 
Pregnancies. At each census, we will ask whether any household 
members are pregnant, and at the follow-up census, inquire about the 
status of the fetus, recording the date of births and deaths. 
• 
Dates of Births, Deaths, and Migrations. These dates will be obtained in 2 
ways. First, all households will be asked whether any children were born 
since the previous census, including any infants who already have died, 
and dates will be recorded. Census workers will also document the date 
that new households moved into the community, and will ask whether 
any children were born or have died since making the move. If a child is 
discovered to be deceased but did not appear on any previous census, the 
census worker will ask the next of kin about the birthdate, and for how 


 
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Dr. Tom Lietman 
36 | P a g e  
 
long the child lived in the community being censused. Second, the key 
informant system will record dates of all births, deaths, and migrations 
among children aged 1-60 months, and will report these data to the verbal 
autopsy interviewer. The verbal autopsy interviewer will enter these data 
into a separate section of the electronic data capture system. 
Chapter 11: Training  
 
11.1 
Standardization 
The three collaborating institutions – UCSF, JHU, and LSHTM – will work together 
prior to MORDOR 0 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. 
11.2 
Pilot Studies 
Prior to MORDOR 0, non-study communities at each study site will be randomly 
selected as “pilot” villages from the same pool as the mortality and morbidity 
communities. The research team will conduct all of the study procedures as 
described in the mortality study and morbidity study (census, sample collection, 
and treatment) in these pilot villages for training purposes. Similar to the 
morbidity and resistance communities, samples will be collected from 40 
children, aged 1 – 60 months and 40 children, aged 7 – 12 years. For morbidity, 
blood samples (thick smears, microcuvettes, dried blood spots), conjunctival 
swabs, and anthropometric measurements (height, weight, MUAC) will be 
collected. For resistance, nasopharyngeal, nares swabs, and stool specimens will 
be collected. Consent will be obtained from the village chiefs and the parents/ 
guardians of each child prior to sample collection. Everyone in pilot communities 
will receive one dose of oral azithromycin after sample collection. If the results of 
the trial are positive, everyone in these villages will also be treated with mass 
oral azithromycin at the end of the study. 
 
 
Chapter 12: Sample Organization, Transport, and Storage 
 


 
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Dr. Tom Lietman 
37 | P a g e  
 
12.1 
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 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. 
 
12.2 
Specimen Transport  
After sample collection, samples from the field will be transported to a local 
health facility or research center at each study site. 
 
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 a local 
laboratory (depending on capacity at each study site). 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.  
 
Clinic-based Swabs (non-core) 
NP swabs in STGG media will be stored in the -20°C freezer at the health 
clinic, and then transported for processing. NP swabs in DNA/RNA 
Shield media will be stored in ambient temperature at the health clinic, 
then transported for processing. 
 
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 -70°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  


 
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Dr. Tom Lietman 
38 | P a g e  
 
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 
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.  
 
12.3 
Specimen Storage 
Sample storage will occur in two stages: short-term and long-term  
 
12.3.1 Short-term Sample Storage 
Samples will be labeled with study ID only and are unidentifiable without access 
to the study database. Lab capacity and sample export regulations vary between 
study sites. If there is capacity to perform sample processing in a quality manner 
(as dictated by IRBs in Tanzania and Malawi), samples will be processed in-
country. Otherwise, samples will be shipped to UCSF, JHU, or LSHTM to 
process core samples and any secondary processing. 
 
12.3.2 Long-term Sample Storage 
Depending on each study site’s sample export regulations, allowable de-
identified samples from Malawi, Niger, and Tanzania 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 investigators and other interested parties.    
 
12.4 
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. 
 
Chapter 13: Microbiology and Molecular Laboratory Procedures 
Participant names will not be included in laboratory records and samples will be 
de-identified.  Therefore, laboratory personnel will be masked, preventing 
identification of the individuals infected.  Lab results will not be available for 
weeks, if not months.  Thus, all individuals will be treated according to their 
study arm, whether or not their lab tests reveal evidence of infection. 
 
13.1 
Storage and Transport Media 
 
13.1.1 Preparation of STGG Transport Medium 
Intended Use 


 
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Manual of Operations and Procedures 
 
Dr. Tom Lietman 
39 | P a g e  
 
STGG was originally used for long term storage of bacteria at -70C. This media 
has also been used to successfully transport and recover bacteria from specimens 
collected on swabs.   
 
Formula 
 
Procedure 
 
1. Combine four ingredients in flask and swirl to dissolve (about ½ 
hour).  Use glass cylinder to measure Glycerol.  Dispense solution 
in 1.0ml amounts into screw-cap 1.5ml vials (Sarstedt Microtube, 
Newton, N.C.) Use Sarstedt rack for easy dispensing. 
2. Autoclave vials with caps screwed on loosely at 15 lb/in2 and 121 
C for 10 minutes.  
3. Do not autoclave vials in plastic storage boxes (boxes will not close 
when cooled). Use old bottom from broken boxes. 
4. STGG should appear milky white to light tan in color with a 
precipitate at the bottom. 
 
Storage and Shelf Life 
Cool vials after autoclaving, screw caps tightly and store at -20 or 4C until use. 
Use vials within 6 months of preparation. 
 
13.1.2 Preparation of Zymo DNA/RNA ShieldTM 
 
Manufacturer’s Product Description 
DNA/RNA ShieldTM ensures nucleic acid stability during sample 
storage/transport at ambient temperatures, preserving the genetic integrity and 
expression profiles of samples. There is no need for refrigeration or specialized 
 
Ingredients per number of 1 ml vials 
Ingredients 
1 
50  
100  
200 
300 
400 
500           1000 
Skim milk powder 
(Difco, Detroit, Mich.) 
Cat.232100 500g 
.02g 
1g 
2.0g 
4g 
6g 
8g 
10g              20 
Tryptone soy broth 
(Oxoid-CM129 500g) 
Fisher cmo129 
.03g 
1.5g 
3.0g 
6g 
9g 
12g 
15g              30g 
Glucose (Difco-
Dextrose cat 215530, 
500g)   
.005g 
.25g 
.5g 
1g 
1.5g 
2g 
2.5g               5g 
Glycerol (Difco- cat 
228220 500g) 
.1ml 
5 
10ml 
20ml 
30ml 
40ml 
50ml        100ml 
 
Distilled water 
1ml 
50ml 
100ml 
200ml 
300ml 400ml 
500ml    1000ml 


 
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Dr. Tom Lietman 
40 | P a g e  
 
equipment. DNA/RNA ShieldTM effectively lyses cells and inactivates nucleases 
and infectious agents (virus), and it is compatible with various collection and 
storage devices (vacuum tubes, swabs, nasal, buccal, fecal, etc.). 
 
Materials Required 
 
Product 
Supplier 
Catalog No. 
DNA/RNA ShieldTM 
1X solution 
Zymo Research 
Corp. 
R1100-50 (50 ml) 
R1100-250 (250 ml) 
2ml Loop Screw Cap Micro Tube, PCR 
Performance Tested 
(DNA-, DNase-, RNase-, & PCR inhibitor-free) 
Sarstedt Inc. 
72.694.416 
Insert for Loop Screw Cap, Violet 
Sarstedt Inc. 
65.713.007 
 
Procedure 
1. Prepare tubes by adding violet cap inserts to the top of each tube (these 
are for color coding purposes to help distinguish the DNA/RNA Shield 
solution from other media being used in the field). 
2. Before aliquoting the DNA/RNA Shield, gently mix the solution by either 
swirling it or inverting the bottle a few times (trying not to create too 
many bubbles) to ensure that the solution is homogenous. In a clean 
biosafety cabinet, use sterile technique to aliquot 1 ml of the solution into 
each 2 ml tube. 
3. When you have finished aliquoting, make sure that the cap on each tube 
has been tightened to prevent tubes from leaking during transport. 
 
Storage and Shelf Life 
Store prepared tubes at room temperature (20°C-25°C) until use; do not freeze 
solution prior to collecting samples. Zymo guarantees the shelf-life of 
DNA/RNA Shield TM up to 1 year from the date of receipt. Therefore, if the 
solution is aliquoted right away, then prepared tubes should be good for 1 year. 
The shelf-life may be longer, depending on storage conditions. 
 
13.1.3 Preparation of Amies Transport Medium 
Rectal swabs will be collected and transported in this medium. It is best prepared 
from ready to use dehydrated powder, available from most suppliers of culture 
media. It is usually used at a concentration of 2 grams in every 100 ml distilled 
water. Fisher Scientific has a dry powder that’s available and can be used to 
prepare the media in house. 
 
Procedure 


 
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Manual of Operations and Procedures 
 
Dr. Tom Lietman 
41 | P a g e  
 
1. Prepare as instructed by the manufacturer. Dispense the well-mixed 
medium into screw-cap tubes. Sterilize by autoclaving (with caps 
loosened) at 121°C for 15 minutes. 
2. When the medium has cooled, tighten the caps. During cooling, invert the 
tubes to ensure an even distribution of the charcoal. 
 
Storage and Shelf Life 
Prepared Amies medium in tightly screw-capped tubes can be stored in a cool 
place at room temperature (away from direct light) until use. Refer to the 
manufacturer’s instructions/guidelines for specifics on expiration dates.  
 
13.1.4 Preparation of Nutrient Agar Medium/Slants 
For isolate storage and shipping, blood agar base (BAB), tryptone soy agar (TSA), 
heart infusion agar (HIA) and nutrient agar are examples of good storage media 
for enteric organisms. Nutrient agar slants will be used to store/ship isolates 
originating from samples such as the rectal swabs. Nutrient agar medium is best 
prepared from ready to use dehydrated powder, available from most suppliers 
of culture media (i.e. Fisher Scientific). In general, nutrient agar is used at a 
concentration of 2.8 g in every 100 ml distilled water. 
 
Procedure 
1. Prepare as instructed by the manufacturer. Dispense the well-mixed, pre-
melted agar medium into tubes (anywhere from 3-5 ml, depending on the 
size of the tube). Sterilize by autoclaving the tubes (with caps loosened) at 
121°C for 15 minutes. 
2. While the tubes are still hot after autoclaving, place them in a slanted 
position to provide a short slant and deep butt (2 to 3 cm). Once medium 
is cooled and agar is set, caps can be tightened and slants should be ready 
for use. 
 
*To inoculate, use an inoculating loop to streak the isolate onto the surface of the 
agar slant. Incubate overnight at 35° to 37°C.  
 
Storage and Shelf Life 
Prepared nutrient agar slants can be stored in a cool dark place and may have a 
shelf life of up to 2 years provided there is no change in the appearance of the 
medium to suggest contamination or deterioration. Refer to the manufacturer’s 
instructions/guidelines for specifics on expiration dates. 
 
13.2 
Methods for Malaria Testing 
 
13.2.1 Blood Smears 


 
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Thin and thick blood smears will be read using standard microbiological 
techniques at local laboratories at each study site. 
 
13.2.2 Dried Blood Spots 
 
Extracting Genomic DNA from Dried Blood Spots 
 
1. Remove FTA card from plastic bag - there should be 5 dried blood 
spots. Using the Harris Micro-Punch and mat, punch a 3 mm disc out 
from one of the blood spots. Place the disc into a 1.5 ml 
microcentrifuge tube. *When processing multiple samples at a time, make 
sure to follow the cleaning protocol below in between punching samples. 
2. Add 500 ul of sterile water to the tube and vortex 5 times. 
3. Use a clean pipette tip to transfer the washed disc to a clean 1.5 ml 
microcentrifuge tube. 
4. Add 30 ul of sterile water to the tube with the disc and place it in a 
heating block set to 95°C for 30 min. After incubating the disc, pulse 
vortex the tube 60 times and then centrifuge. Remove the pelleted disc 
with a pipet tip and discard. 
5. Store eluted DNA at -20°C until ready for use. It is best to run PCR as 
soon as possible after extracting DNA. Use 5 ul of eluted DNA in CytB 
PCR reactions (50 ul reaction) as outlined below. 
 


 
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Punch Cleaning Protocol 
1. In between each sample, swirl the Harris Micro-Punch in a beaker 
containing a 1:10 bleach solution for a few seconds and then swirl the 
tip in a beaker containing a 70% ethanol solution. Dry/blot the tip on a 
clean paper towel and then allow the punch to air dry for a couple of 
seconds before using. Discard the used paper towel in the biohazard 
waste. 
a. Blank control - After cleaning the punch, you may want to take 
a blank punch from a clean FTA Elute card with no sample and 
use it as a negative control to confirm that there is no cross 
contamination. 
2. Clean the cutting mat with a Mini Hypewipe (bleach wipe) and then 
wipe down with ethanol solution using a paper towel. Discard bleach 
wipe and paper towel in the biohazard waste. (You only have to clean 
the mat every couple of samples, as long as you use different areas of 
the mat to punch out each sample – for example, you can clean the mat 
after every 4 samples if the samples are punched out in four different 
quadrants of the mat). 
 
Amplifying Gene Regions (PCR) 
1. Set up first round of PCR with outer primers: Protocol was adapted 
from Hsiang, et al. & Steenkeste, et al. papers40,45 and based on 
guidelines provided for Qiagen TopTaq DNA Polymerase (refer to 
Qiagen TopTaq PCR Handbook).46 
 
Gene 
Primer 
Sequence 
CytB 
Outer 
Forward 
CGGTCGCGTCCGGTAGCGTCTAATGCCTAGACGTATTCCTGATTATCCAG 
Outer 
Reverse 
CGCATCACCTCTGGGCCGCGTGTTTGCTTGGGAGCTGTAATCATAATGTG 
Inner 
Forward 
GAGAATTATGGAGTGGATGGTG 
Inner 
Reverse 
TGGTAATTGACATCCAATCC 
 
TopTaq PCR Buffer (10X)  
5 ul 
dNTPs (10 mM) 
 
 
1 ul 
Forward Primer (10 uM) 
 
2 ul 
Reverse Primer (10 uM) 
 
2 ul 
Template DNA 
 
 
5 ul 
10x CoralLoad Dye  
 
5 ul 
Taq Polymerase 
 
          0.25 ul 
Water  
 
                    29.75 ul 
 
 
Total Volume: 
 
          50 ul 
 


 
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Note: When setting up several PCR reactions, make up a mastermix 
for each primer set by multiplying each volume by the number of 
reactions you have (making up 2-3 more reactions than needed). 
Aliquot out 45 ul of mastermix for each reaction and then add 5 ul 
of template DNA for each sample.   
 
• Use the following cycling conditions for the Bio-Rad S1000 
thermocycler: 
Initial step: 95°C for 5 min 
*Denature: 94°C for 30 sec 
Anneal: 60°C for 90 sec 
Extend: 72°C for 90 sec 
Repeat from * (Denaturing step to extension step) for 40 cycles 
Final step: 72°C for 10 min 
 
2. Set up second round of PCR with inner primers: 
a. Use the same recipe as above, but use the specific set of inner 
primers instead of the outer primers for each reaction. Also, use 
5 ul of amplicon DNA (DNA from first PCR reaction) as your 
target. 
b. Use the same cycling conditions as above. 
 
Detecting PCR Products 
Use gel electrophoresis to detect and verify the size of expected PCR products. 
 
If running a single-comb 2% agarose e-gel (12 samples total, including 
MW marker): 
1. Load each well that you plan to add a sample to with 15 ul of water. 
Then load 5 ul of each sample that contains your PCR product into 
the correct wells. Load 20 ul of water into any remaining empty 
wells. 
2. Load 15 ul of water into the well designated for the MW marker, 
and then add 5 ul of the marker to that well. 
3. The single-comb 2% e-gel needs to run for about 30 min. 
 
If running a double-comb 2% agarose e-gel (16 samples total, plus 2 MW 
markers): 
1. Load each well that you plan to add a sample to with 15 ul of water. 
Then load 5 ul of each sample that contains your PCR product into 
the correct wells. Load 20 ul of water into any remaining empty 
wells. 
2. Load 5 ul of water into the well designated for the MW marker, and 
then add 5 ul of the marker to that well. 


 
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3. The double-comb 2% e-gel needs to run for about 15 min. 
 
If running a 48 2% agarose e-gel (48 samples, plus 4 MW markers):  
1. Load each well that you plan to add a sample to with 10 ul of water. 
Then load 5 ul of each sample that contains your PCR product into 
the correct wells. Load 15 ul of water into any remaining empty 
wells. 
2. Load 10 ul of water into the well designated for the MW marker, 
and then add 5 ul of the marker to that well. 
3. The 48 2% e-gel needs to run for about 20 min. on program “EG”. 
 
(For e-gel procedures: see technical manual for running the gel on the Mother 
E-Base – Do not do a pre-run, but remember to set up the program for the E-
base and to place the gel in the E-Base before loading the samples in the gel) 
 
Purifying PCR Products of Positive Samples 
Use Qiagen QIAquick PCR Purification kit and corresponding protocol, eluting 
the DNA in 50 ul of water.47,48  
 


 
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Procedure 
 
1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. In our case 
transfer whatever is left of the PCR sample (about 45 ul) to a clean 1.5 ml 
microcentrifuge tube and add 225 ul of Buffer PB to it. 
 
2. If pH indicator I has been added to Buffer PB, check that the color of the 
mixture is yellow.  
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 
5.0, and mix. The color of the mixture will turn to yellow. 
 
3. Place a QIAquick spin column in a provided 2 ml collection tube. 
 
4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–
60 s. 
 
5. Discard flow-through. Place the QIAquick column back into the same tube. 
 
6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–
60 s. 
 
7. Discard flow-through and place the QIAquick column back in the same tube. 
Centrifuge the column for an additional 1 min. IMPORTANT: Residual ethanol 
from Buffer PE will not be completely removed unless the flow-through is 
discarded before this additional centrifugation. 
 
8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube. 
 
9. To elute DNA, add 50 µl water (pH 7.0–8.5) to the center of the QIAquick 
membrane and centrifuge the column for 1 min. Alternatively, for increased 
DNA concentration, add 30 µl water to the center of the QIAquick membrane, 
let the column stand for 1 min, and then centrifuge. IMPORTANT: Ensure that 
the water is dispensed directly onto the QIAquick membrane for complete elution 
of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer 
volume, and 28 µl from 30 µl elution buffer. Elution efficiency is dependent on 
pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When 
using water, make sure that the pH value is within this range, and store DNA at –
20°C as DNA may degrade in the absence of a buffering agent. 
 


 
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Speciation via Sequencing 
Sequetech will automatically quantify the amount of DNA that we send them, 
and, based on their readings, they will adjust the DNA concentration and 
optimize the sequencing reaction. For a PCR product that is about 800 bp, they 
only use about 4 ng of DNA for each sequencing reaction. All the primers that 
they use in their sequencing reactions are at a concentration of 1.6 uM and they 
only need 2 ul per reaction. 
 
Preparing the samples for Sequetech 
Note: Send primers and template DNA (your samples) in separate tubes – if you 
premix them, then they cannot quantify your DNA accurately and cannot 
optimize the sequencing reaction. Also, make sure that all tubes are labeled 
clearly and match the names that you enter on the order form. 
 
1. Aliquot 6 - 10 ul of each of the purified PCR product that you want to 
have sequenced into a 1.5 ml microcentrifuge tube and label it clearly 
(this should be enough DNA for them to sequence each sample both 
ways). 
2. Make up each nested primer (keep the forward and reverse primer in 
separate tubes) that corresponds to your PCR product at a 
concentration of 3 uM. To do this take 3 ul of the 100 uM stock solution 
and add it to 97 ul of TE (this will give you a total volume of 100 ul). 
Based on the number of sequencing reactions you are ordering, 
calculate how much of the 3 uM primer solution you need to aliquot 
and send. 
*They will need approximately 1.07 ul of the 3 uM primer solution for 
each reaction, so to be on the safe side, allocate 2 ul of the 3 uM 
primer solution for each sequencing reaction. 
Calculation: amount of primer needed = (# of sequencing reactions 
using primer) x 2 
3. Organize all the aliquoted and labeled tubes in a storage box and then 
place the storage box in a sample bag. 
4. Place the sequencing order online through Sequetech’s website 
http://www.sequetech.com/, making sure to list all your template 
DNAs’ names, primers’ names, the concentration that your primers are 
at (3 uM), and the approximate size of your PCR products. Print out 
the order form once you have finished and place a copy of it in the bag 
with your samples. 
5. Schedule a pick-up directly from your location (make sure that 
everything is ready and scheduled before 3 pm). 
6. You can check results online within 24 hours. 


 
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13.3 
Methods for Resistance Testing 
 
13.3.1 Isolation & Identification of Organisms Using Microbiological 
Techniques 
Various organisms from nasal, nasopharyngeal, and rectal swabs will be isolated 
using the media and conditions in Table 2 and identified using the methods 
summarized in Table 3 and below. Standard microbiological handbooks should 
be referenced for more detailed information. 
 
Procedure for Masking Samples 
The microbiologist and all lab staff will be masked to the community of origin. 
 
Laboratory Results Reporting 
All lab results will be kept in computer files as well as in hard-copy form by the 
Microbiologist. 
 
Table 2. Isolation of Organisms from Source - Primary Plating Media & 
Incubating Conditions 
 
Source 
Media 
Incubation 
Organisms Isolated 
 Nasal Swab 
Blood and MSA 
35°C 
5% CO2 
S. aureus (core) 
Nasopharyngeal 
Swab 
Blood and Strep 
select plate, 
chocolate, chocolate 
with bacitracin 
35°C 
5% CO2 
S. pneumoniae (core) 
H. influenzae (non-core) 
N. meningitidis (non-core)  
Rectal Swab 
Blood and 
MacConkey 
35°C 
E. coli (core) 
 


 
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Table 3. Identification of Organisms 
 
Organism 
Stain 
Identification Tests 
S. aureus 
(core) 
Gram stain 
Catalase and Tube Coagulase or latex 
agglutination reagent 
S. pneumoniae 
(core) 
Gram stain 
Catalase, Optochin disc and/or Desoxycholate 
reagent, alpha-hemolytic on blood agar 
H. influenzae 
(non-core) 
Gram stain 
Satelliting growth on blood agar, ALA or other 
identification tests 
N. meningitidis 
(non-core) 
Gram stain 
Sugars utilization or other identification kit 
S. pyogenes 
(core) 
Gram stain 
Catalase, beta-hemolytic on blood agar, and 
Bacitracin disc or PYR test 
E. coli 
(core) 
Gram stain 
Indole, oxidase, beta-hemolytic on blood agar, 
PYR and other identification tests as required 
 
Description of Identification Tests: 
We will follow the standard quality control methods already in place at the 
laboratory. For example, lab staff will perform positive and negative growth 
controls for all media, and positive controls for all stains and identification tests 
at a pre-determined schedule. 
 
Catalase Test: 
A few drops of 3% hydrogen peroxide are added to colonies removed from agar 
and smeared onto a clean glass slide, and immediately observed for the release of 
oxygen or “bubbling.” Bubbling is interpreted as the presence of catalase. Lack of 
bubbling is a negative test. 
 
Tube Coagulase: 
Coagulase plasma is inoculated with an isolated staphylococcal colony and 
incubated for up to 24 hours at 37°C. Any degree of clot formation is a positive 
reaction and no clot is a negative reaction. 
 
Latex Agglutination: 
A few staphylococcal colonies are mixed with the latex agglutination reagent and 
observed for agglutination. A positive reaction is agglutination of the latex 
particles, while no agglutination is a negative reaction. 
 
Optochin Test: 
An optochin disc is placed onto a blood agar subcultured plate that has been 
inoculated with a pure culture of the test organism. Plates are incubated for 24 
hours at 35°C in CO2. If a zone of inhibition of 14 mm or more is formed, then it’s 
S. pneumoniae. 


 
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Desoxycholate (10%) Test: 
A drop of reagent is added to a well-isolated colony on a blood agar plate. 
Colonies of S. pneumoniae will disintegrate after 30 minutes. 
 
Satelliting Growth on Blood Agar: 
Colonies resembling Haemophilus are subcultured to a non-selective blood agar 
plate and cross streaked with Coagulase negative Staphylococcus. Plates are 
incubated in CO2 overnight and examined for satellite growth around 
Staphylococcus. 
 
ALA Test: 
 
Sugars Utilization Tests: 
 
Indole Test: 
 
Oxidase Test: 
 
PYR Test: 
 
13.3.2 Microbiological Resistance Testing for Swabs 
Resistance testing will be performed using the Kirby Bauer disc diffusion assay 
or E-test strips (to determine MIC’s). 
 
Quality Control 
E. coli ATCC 25922, S. pneumoniae ATCC 49619, H. influenzae ATCC 49247, S. 
aureus ATCC 25923 and 29213 are used for quality control testing of antibiotic 
discs and E-test strips. 
 
Kirby Bauer Assay 
A suspension of the isolate is prepared and turbidity adjusted until it reaches the 
0.5 McFarland standard. The suspension is used to inoculate the appropriate 
susceptibility agar plate for confluent growth. The plate is allowed to dry. 
Antimicrobial disks are placed on the agar plates with sterile forceps or tweezers; 
disks may not be moved once they have touched the plates. After the disks are 
placed on the plate, the plates are inverted and incubated at 35°C for 16 to 18 
hours. After incubation, the diameter of the zones of complete inhibition 
(including the diameter of the disk) (Figure 2) are measured and recorded in 
millimeters. The measurements can be made with a ruler or sliding caliper on the 
undersurface of the plate without opening the lid. The zones of growth inhibition 
will be compared with the zone-size interpretative table and recorded as 


 
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susceptible, intermediate, or resistant to each drug tested according to CLSI 
guidelines (see Appendix). 
 
 
Figure 2. Results of the disk diffusion assay. This Shigella isolate is resistant to 
trimethoprim-sulfamethoxazole and is growing up to the disk (SXT), the zone of 
which is recorded as 6 mm. 
 
E-test strips. 
In addition, azithromycin Minimum Inhibitory Concentrations (MIC) are 
determined using E-test strips. A suspension of the isolate is prepared and 
turbidity adjusted until it reaches the 0.5 McFarland standard. The suspension is 
used to inoculate the appropriate susceptibility agar plate for confluent growth. 
The plate is allowed to dry. E-test strips are placed on the agar plates with sterile 
forceps or tweezers; strips may not be moved once they have touched the plates. 
After the strips are placed on the plate, the plates are inverted and incubated at 
35°C for 20 to 24 hours. The MIC is read where the ellipse intersects the MIC 
scale on the strip. The MIC will be compared with the interpretative table 
according to CLSI guidelines (see Appendix).  
 


 
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Table 4. Antibiotics Testing Guidelines  
 
Specimen 
Organism 
Antibiotics Tested 
Nasal 
S. aureus 
Azithromycin, cefoxitin 
Nasopharyngeal S. pneumoniae 
H. influenzae 
N. meningitidis 
Azithromycin, clindamycin, penicillin 
(Note: S. pneumoniae - 1 ug oxacillin used to 
predict penicillin resistance for Kirby Bauer)  
Stool 
E. coli 
Azithromycin, ampicillin, co-trimoxazole 
 
Materials Required for Susceptibility Testing: 
• MH w/ 5% sheep blood plates 
• MHA plates 
• MH broth or sterile 0.85% saline 
• Sterile cotton-tipped swabs 
• 0.5 MacFarland Standard 
• Blood and chocolate plates 
• Caliper for reading zones 
• Forceps  
• Light source 
• Vortex 
• 35°C ambient air and 35°C CO2 incubator  
 
13.3.3 Nucleic Acid Based Resistance Testing for Stool Specimens 
Stool specimens not cultured will undergo nucleic acid based resistance testing. 
DNA from the stool samples will be extracted using some type of DNA isolation 
kit (i.e. Norgen Stool DNA Isolation Kit) and the appropriate corresponding 
protocol.49 PCR and gel electrophoresis will then be performed with specific 
primers to detect genes associated with E. coli and antibiotic resistance 
(specifically macrolide resistance). 
 


 
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Extracting DNA from Stool Procedure 
1. Lysate Preparation 
a. 
Mix the sample well to make sure that the stool/preservative mixture is homogenous. Carefully 
remove the lid from the collection tube and aliquot 200 μl of the stool/preservative liquid and add it to 
a Bead Tube provided with the kit (it may be easier to use a pipet tip that has been cut to make the 
opening wider or to use a large orifice pipet tip). 
b. Add Lysis Solution to the Bead Tube, so that the final volume is 1 ml (for our purposes, add 800 μl of 
Lysis Solution). Vortex and centrifuge briefly. 
c. 
Add 100 μl of Lysis Additive and vortex briefly. 
d. Secure the Bead Tube horizontally on a flat-bed vortex pad with tape, or secure the tube in any 
commercially available bead beater equipment (e.g. Scientific Industries’ Disruptor GenieTM). Vortex 
for 3 minute at maximum speed. 
e. 
Centrifuge the tube for 2 minutes at 14000 × g (~14,000 RPM). 
f. 
Transfer up to 600 μl of supernatant to a DNAase-free microcentrifuge tube (not provided). 
g. 
Add 200 μl of Binding Solution, mix by inverting the tube a few times, and incubate for 10 minutes on 
ice. 
h. Spin the lysate for 2 minutes to pellet any cell debris. 
i. 
Using a pipet, transfer up to 700 μl of supernatant (avoid contacting the pellet with the pipet tip) into a 
2 ml DNAase-free microcentrifuge tube (not provided). 
j. 
Add an equal volume of 70% ethanol (provided by the user) to the lysate collected above (100 μl of 
ethanol is added to every 100 μl of lysate). Vortex to mix. 
 
2. Binding to Column 
a. 
Assemble a spin column with one of the provided collection tubes. 
b. Apply 600 μL of the clarified lysate with ethanol onto the column and centrifuge for 1 minute at 14000 
× g (~14,000 RPM). Discard the flow through and reassemble the spin column with the collection tube. 
Note: Ensure the entire lysate volume has passed through into the collection tube by inspecting the 
column. If the entire lysate volume has not passed, spin for an additional minute. 
c. 
Repeat step 2b with the remaining volume of lysate mixture. 
 
3. Column Wash 
a. 
Apply 500 μL of Wash Solution I to the column and centrifuge for 1 minute. 
Note: Ensure the entire wash solution has passed through into the collection tube by inspecting the 
column. If the entire wash volume has not passed, spin for an additional minute. 
b. Discard the flow through and reassemble the spin column with its collection tube. 
c. 
Apply 500 μL of Wash Solution II to the column and centrifuge for 1 minute. 
d. Discard the flow through and reassemble the spin column with its collection tube. 
e. 
Repeat 3c and 3d. 
f. 
Spin the column for 2 minutes in order to thoroughly dry the resin. Discard the collection tube. 
 
4. DNA Elution 
a. 
Place the column into a fresh 1.7 mL Elution tube provided with the kit. 
b. Add 50 μL of Elution Buffer to the column. 
c. 
Centrifuge for 2 minutes at 200 x g (~2,000 RPM), followed by a 1 minute spin at 14,000 x g (~14,000 
RPM). Note the volume eluted from the column. If the entire volume has not been eluted, spin the 
column at 14,000 x g (~14,000 RPM) for 1 additional minute. 
d. (Optional): An additional elution may be performed if desired by repeating steps 4b and 4c using 50 
μL of Elution Buffer. The total yield can be improved by an additional 20-30% when this second 
elution is performed. 
 
5. Storage of DNA 
The purified genomic DNA can be stored at 2-8°C for a few days. For longer term storage, -20°C is 
recommended. 
 


 
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Amplifying Gene Regions 
We will use DNA extracted from stool specimens and published primers to perform 
PCR reactions for the genes of interest:50-52 
 
Purpose 
Genes 
Detecting E. coli 
malK (encodes for malB 
promoter) 53 
 
*This gene will detect for both E. 
coli and Shigella since the two are 
closely related and are difficult to 
discriminate from one another. 
 
Detecting Antibiotic 
Resistance 
mph (A, B) 
erm (A, B, C) 
mef (A) 
ere (A, B) 
msr (A) 
 
 
13.4 
Methods for Chlamydia trachomatis  
 
13.4.1 Description of Abbott RealTime Assay 
Swabs will be processed with the Abbott RealTime assay for C. trachomatis, using 
the automated Abbott m2000 System. The RealTime assay targets the cryptic 
plasmid of C. trachomatis. The assay has been shown to be highly sensitive and 
specific for the diagnosis of sexually transmitted C. trachomatis, with sensitivities 
exceeding that of the nucleic acid amplification test used in most recent trachoma 
studies (Roche AMPLICOR).54,55  
 
13.4.2 Processing of Samples for PCR 
Samples are handled as per Abbott RealTime sample processing protocol, with 
the following modifications: 
1. Samples are boiled for 10 minutes at 100°C.  Boiling of samples is an 
accepted treatment method to remove substances that may be inhibitory 
to the PCR amplification process 
2. Samples are pooled. 
 
13.4.3 Procedure for Masking Samples 
In order to mask the location origin, the control status, and the clinical exam 
grading of the conjunctival samples collected in the field, the Database Manager 
will assign an identification number different from the random number assigned 
to each child. The PCR results will be recorded according to this laboratory 
identification number, thus masking the lab until all the samples have been 


 
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processed. The Database Manager will then link the lab ID number to the 
random sample number to reveal the test results by village. 
 
13.4.4 Procedure for Pooling Samples 
We will increase the efficiency of chlamydial testing by combining swabs from 
the same age stratum and same community into pools of 5 random swabs for 
processing. An internal control will be run with each pool to rule out the 
possibility of PCR inhibitors. Any inhibitory pools will be re-tested, and if still 
inhibitory, the swabs will be individually re-tested. If PCR from any pool is 
equivocal, then all swabs from the pool will be tested individually. While 
samples will necessarily be diluted in this process, this is not thought to affect the 
sensitivity of the test.56 We will unpool all positive pools in the 0-5 year age 
stratum and estimate the community prevalence of chlamydial infection as the 
proportion of positive swabs. We will estimate the community prevalence of 
infection in the 6-9 and ≥10 year age strata using maximum likelihood 
estimation, similar to our previous trials: the number of individual swabs with 
the maximum likelihood of having resulted in the observed pooled results will 
be chosen as the estimate for that village.57,58 
 
In order to pool the conjunctival samples in the lab, the microbiology lab staff 
will assign a new pool ID number for every sample, and samples will be stored 
at -80°C freezer until PCR testing (if not processed that day). 
 
13.4.5 Quality Control 
1. A C. trachomatis(+) control and a C. trachomatis(-) control (targeting 136 
base pairs of a pumpkin gene) is included in each test run of the Abbott 
RealTime assay. 
2. To test the effect of sample processing, a known positive sample is 
processed and tested in each test run.  (This control is helpful when testing 
large numbers of negative samples.) 
3. An internal control intended to identify specimens that contain 
polymerase inhibitor is run routinely on each sample.  The internal control 
helps identify false negative results. 
 
13.4.6 Quantification 
For every 0-5 year old who tests positive for chlamydia, we will also run PCR for 
the beta actin gene on the same sample, in order to normalize the quantity of 
chlamydial DNA to the amount of the specimen. Quantitative results from the 
Abbott system are given in terms of a decision cycle (DC) number. We will 
generate a ratio of the DC number of the chlamydial DNA to the DC number of 
the beta actin gene and use the resulting ratio as the chlamydial load.  
 


 
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Chapter 14: Study Medication 
Children aged 1-60 months on the current census will be offered weight- or 
height-based, directly observed, oral suspension (azithromycin or placebo) every 
6 months for 2 years (as performed in trachoma programs) at each study site. 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. 
 
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. 
 
14.1 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. 
 
14.1 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. 
 
14.2 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 each study site will manage the customs process and 
transport the medication from the port to storage sites.   
 


 
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14.3 Medication Quality Control 
Study medication will be stored at each country site prior to use.  The study 
coordinator and other local staff will regularly check and record the study 
medication expiration dates.  The expiration dates on the medication containers 
will be strictly monitored and all expired study medicine will be discarded 
appropriately. 
 
14.4 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 
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 UCSF DCC will identify any children who have missed 2 or more 
consecutive treatments, and forward this information to the site 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. 
 
14.5 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 local program representatives immediately. If, for any reason, 


 
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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.  
 
14.6 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.  
 
14.6.1 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.  
 
14.6.2 Active Adverse Events Monitoring 
 
During Census 
We will employ two forms of active monitoring for adverse events. First, during 
each follow-up census we will ask the guardians of all 7-12 month-old children in 
all study communities (both mortality and morbidity), whether their children 
had an adverse event after the previous mass medication distribution. This will 
allow assessment of adverse events from medication distribution among the 
individuals aged 1-6 months at the time of the distribution. This information will 
be incorporated into the electronic data capture system so that this question is 
asked for any child aged 7-12 months. Note that this method will be subject to 
recall bias, so we will also perform a house-to-house survey (described below). 
 


 
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Infant Adverse Events Survey 
To identify any adverse events associated with mass treatment, the UCSF DCC 
will randomly select 30 study communities (15 azithromycin and 15 control 
communities) 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 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, nausea, diarrhea, 
dyspepsia, constipation, hemorrhoids or rash. To elicit symptoms of serious 
infantile hypertrophic pyloric stenosis which has been reported in rare occasions 
with macrolide use, census workers will be trained to ask about projectile 
vomiting and feeding problems. 
 
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 on-site study coordinator; who must, within 24 hours, submit a Pfizer IIR 
SAE Form to mordor.sae@gmail.com. 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. TL will also forward SAE 
to DSMC if meets criteria of being possibly related to study drug. 
 
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. 
 
14.6.3 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. 
 
14.8  Supply issues  
If a study site runs out of a treatment letter, a request should be sent to 
mordor.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 (TCP, KR, or ZZ) 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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Chapter 15: Protection of Human Subjects 
Before the study begins, each site will obtain formal ethical approval from their 
respective national ethics committee. In addition, local staff will approach 
community leaders to describe the study and answer any questions. Study staff 
will proceed only if local leadership consents to participate. At the individual 
level, we will obtain verbal consent from a parent or guardian for all study 
activities with patient contact, including censuses, examinations, and treatments.  
 
Verbal consent is the best way to obtain consent, due to the high illiteracy rates in 
each study area. Children will be included in the study only following the receipt 
of 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. 
 
15.1 
Institutional Review Board Approval 
 
 
15.1.1. Niger 
 
UCSF Committee on Human Research 
UCSF’s Committee on Human Research will annually review study protocol for 
ethical approval. 
 
Emory University Institutional Review Board 
The Emory University Institutional Review Board will annually review study 
protocol for ethical approval. 
 
Comité d’Ethique du Niger   
The study protocol will be reviewed and granted ethical approval by the Comité 
d’Ethique du Niger before any patient-related research activities begin. 


 
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15.1.2 Tanzania 
 
Johns Hopkins University Institutional Review Board 
The Johns Hopkins University Institutional Review Board will annually review 
study protocol for ethical approval. 
 
Tanzania National Institute for Medical Research Ethical Review Board   
The study protocol will be reviewed and granted ethical approval by the 
Tanzanian IRB before any patient-related research activities begin. 
 
15.1.3 Malawi 
 
London School of Hygiene & Tropical Medicine Institutional Review Board 
The London School of Hygiene & Tropical Medicine Institutional Review Board 
will annually review study protocol for ethical approval. 
 
Malawi IRB 
The study protocol will be reviewed and granted ethical approval by the 
University of Malawi Blantyre IRB before any patient-related research activities 
begin.    
 
15.2 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 Malawi, Niger, and Tanzania prior to study 
implementation. Then they will be read aloud to each study participant (and 
his/her parent/guardian) by a local 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 verbal 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.  
 


 
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15.3 Risks and Benefits of Study Procedures 
 
15.3.1 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. 
   
15.3.2 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. 
  
15.3.3 Stool Collection 
Stool samples have been collected in this setting before, with essentially no risk 
to participants. 
  
15.3.4 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. 
  
15.3.5 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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Chapter 16: Study Monitoring  
The UCSF team will conduct annual monitoring visits to project sites in Malawi 
and Tanzania. The LSHTM team will conduct annual visits to Niger. Annual site 
visits will ensure uniform data collection, as well as adequate training and 
certification. 
Chapter 17: Data and Safety Monitoring Committee Charter 
This Charter is for the Data Safety and Monitoring Committee (DSMC) for 
Mortality Reduction after Oral Azithromycin (MORDOR): OPP1032340.59 
 
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. 
 
17.1 
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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17.2 
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. 
 
Judd Walson, MD, MPH (Chair): pediatrics and global health 
Alan Hightower, MS: biostatistics 
Latha Rajan, MD, MPH&TM: global health 
Emily Anderson, PhD, MPH: bioethics 
Wondu Alemayehu, MD, MPH: global health 
 
17.3 
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.   
 
17.4 
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 


 
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the standard operating procedures and to discuss the format and content of the 
Open and Closed Reports that will be used to present trial results. 
 
The Organizational Meeting will be attended by all DSMC members, lead trial 
investigators, and the trial biostatistician. The DSMC will be given the drafts of 
the trial protocol, the Statistical Analysis Plan, the DSMC Charter, and the 
current version of the case report forms. At subsequent meetings, committee 
members will receive Open and Closed Data Reports. 
 
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. 
 
17.5 
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.  
 


 
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Closed reports, available only to those attending the Closed Sessions of the 
meeting, will include analyses of primary and secondary efficacy endpoints, 
including subgroup and adjusted analyses, AEs and symptom severity, , and 
Open Report analyses that are displayed by intervention group. These Closed 
Reports will be prepared by the study biostatistician.  
 
The Open and Closed Reports should provide information that is accurate, with 
follow-up that is complete to within two months of the date of the DSMC 
meeting. The Reports should be provided to DSMC members approximately 
three days prior to the date of the meeting. 
 
Minutes of the DSMC Meeting 
The research team will prepare minutes for the open portion of the meeting, 
including the DSMC’s recommendations. 
 
Recommendations to the Steering Committee (SC) 
At each meeting of the DSMC during the trial, the committee will make a 
recommendation to the Steering Committee to continue or terminate. This 
recommendation will be based primarily on safety and efficacy considerations 
and will be guided by statistical monitoring guidelines defined in this Charter. 
 
Recommendations to amend the protocol or conduct of the study made by the 
DSMC will be considered and accepted or rejected by the SC. The SC will be 
responsible for deciding whether to continue or to stop the trial based on the 
DSMC recommendations.  
 
The DSMC will be notified of all changes to the protocol or to study conduct. The 
DSMC concurrence will be sought on all substantive recommendations or 
changes to the protocol or study conduct prior to implementation.  
 
The SC may communicate information in the Open Report to the sponsor and 
may inform them of the DSMC recommended alterations to study conduct or 
early trial termination in instances in which the SC has reached a final decision 
agreeing with the recommendation. The SC will maintain confidentiality of all 
information it receives other than that contained in the Open Reports until after 
the trial is completed or until a decision for early termination has been made. 
 
17.6 
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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17.7 
DSMC Contact Information  
 
Table 5: DSMC Contact Information 
Wondu Alemayehu 
Consultant Ophthalmologist 
Berhan Health 
PO Box 6307 
Addis Ababa 
Ethiopia 
+251 910 57 48 42 
walemayehu@berhan-health.org 
Emily Anderson 
Assistant Professor 
Loyola University Chicago 
Health Sciences Division 
2160 S. First Ave. 
Maywood, IL 60153 
708.327.9229 
emanderson@lumc.edu 
Allen Hightower 
Statistician 
2189 Oakawana Drive 
Atlanta, GA 30345 
706.341.0176 
awh1953@gmail.com 
Latha Rajan 
Clinical Associate Professor 
1440 Canal Street 
Suite 2210 
Department of Tropical Medicine 
New Orleans, LA 70112 
504.988.7970 
lrajan@tulane.edu 
Judd Walson, Chair 
Associate Professor 
University of Washington 
Box 359909 
325 Ninth Avenue 
Seattle, WA 98104 
206.744.3695 (office) 
206.612.4571 (mobile) 
walson@uw.edu 
 


 
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17.8 
Contingency DSMC Committee 
 
The MORDOR contingency study delivered open label azithromycin during the 
third year of the study to all participants. At the beginning of year 4, the 
communities were re-randomized to either placebo or azithromycin and a new 
DSMC committee was formed. This committee will follow the same charter as 
the original committee, with the exception of there being no plan to conduct an 
interim analysis and no futility or stopping rules. This DSMC committee is 
comprised of the following individuals:  
Dan Kelly, MD (chair)  
Infectious Disease Specialist,  
Prevention and Public Health Group  
University of California, San Francisco 
San Francisco, CA 94143 
415-476-6224 
Dan.Kelly@ucsf.edu 
Arthur Reingold, MD 
Professor, Epidemiology & 
Biostatistics 
University of California, Berkeley 
2121 Berkeley Way #5419 
Berkeley, CA 94704 
(510) 642-0327 
reingold@berkeley.edu 
 
George Rutherford, MD 
Professor, Epidemiology & 
Biostatistics 
University of California, San Francisco 
San Francisco, CA 94143 
George.Rutherford@ucsf.edu 
 
Chapter 18: Data Collection, Management, and Security 
 
18.1. 
Scope of Data 
Mortality and morbidity data from Malawi, Niger, and Tanzania 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. Costing data will also be collected, including the costs of azithromycin, 
antibiotic distributors, supervisors, drivers, fuel, and time of community volunteers. 
Mortality Study 


 
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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.    
 
Morbidity Study 
Census, mortality, and treatment data collection procedures will be identical to the 
mortality study. In addition, 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, nasal swabs, 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. 
18.2 
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. Each study site will have 
a local data coordinating center within the study area. 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 
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). 


 
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18.3 
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 Salesforce. 
The Salesforce 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 Salesforce 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.  
 
References 
 
1. 
Rajaratnam JK, Marcus JR, Flaxman AD, et al. Neonatal, postneonatal, 
childhood, and under-5 mortality for 187 countries, 1970-2010: a systematic analysis of 
progress towards Millennium Development Goal 4. Lancet 2010;375:1988-2008. 
2. 
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 : 
the journal of the American Medical Association 2009;302:962-8. 
3. 
Shaikh S, Schulze KJ, Kurpad A, et al. Development of bioelectrical impedance 
analysis-based equations for estimation of body composition in postpartum rural 
Bangladeshi women. The British journal of nutrition 2012:1-9. 
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Waage J, Banerji R, Campbell O, et al. The Millennium Development Goals: a 
cross-sectoral analysis and principles for goal setting after 2015 Lancet and London 
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You D, Wardlaw T, Salama P, Jones G. Levels and trends in under-5 mortality, 
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Bhutta ZA, Chopra M, Axelson H, et al. Countdown to 2015 decade report (2000-
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7. 
Black RE, Cousens S, Johnson HL, et al. Global, regional, and national causes of 
child mortality in 2008: a systematic analysis. Lancet 2010;375:1969-87. 


 
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8. 
Arifeen SE, Hoque DM, Akter T, et al. Effect of the Integrated Management of 
Childhood Illness strategy on childhood mortality and nutrition in a rural area in 
Bangladesh: a cluster randomised trial. Lancet 2009;374:393-403. 
9. 
Sazawal S, Black RE. Effect of pneumonia case management on mortality in 
neonates, infants, and preschool children: a meta-analysis of community-based trials. 
Lancet Infect Dis 2003;3:547-56. 
10. 
Moonen B, Cohen JM, Snow RW, et al. Operational strategies to achieve and 
maintain malaria elimination. Lancet 2010;376:1592-603. 
11. 
Solomon A, Zondervan M, Kuper H, Buchan J, Mabey D, Foster A. Trachoma 
control: a guide for programme managers. Geneva: World Health Organization; 2006. 
12. 
Melese M, Alemayehu W, Lakew T, et al. Comparison of annual and biannual 
mass antibiotic administration for elimination of infectious trachoma. JAMA 
2008;299:778-84. 
13. 
Lietman T, Porco T, Dawson C, Blower S. Global elimination of trachoma: how 
frequently should we administer mass chemotherapy? Nat Med 1999;5:572-6. 
14. 
Schachter J, West SK, Mabey D, et al. Azithromycin in control of trachoma. 
Lancet 1999;354:630-5. 
15. 
Bailey RL, Arullendran P, Whittle HC, Mabey DC. Randomised controlled trial 
of single-dose azithromycin in treatment of trachoma. Lancet 1993;342:453-6. 
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Coles CL, Seidman JC, Levens J, Mkocha H, Munoz B, West S. Association of 
mass treatment with azithromycin in trachoma-endemic communities with short-term 
reduced risk of diarrhea in young children. The American journal of tropical medicine 
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Lode H, Borner K, Koeppe P, Schaberg T. Azithromycin--review of key 
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Miller GY, Algozin, K. A., McNamara, P. E. and Bush, E. J,. Productivity and 
economic impacts of feed-grade antibiotic use in pork production. J Agr Appl Econ 
2003;12:469-82. 
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Fry AM, Jha HC, Lietman TM, et al. Adverse and beneficial secondary effects of 
mass treatment with azithromycin to eliminate blindness due to trachoma in Nepal. Clin 
Infect Dis 2002;35:395-402. 
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Sadiq ST, Glasgow KW, Drakeley CJ, et al. Effects of azithromycin on 
malariometric indices in The Gambia. Lancet 1995;346:881-2. 
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Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ. Global and regional 
burden of disease and risk factors, 2001: systematic analysis of population health data. 
Lancet 2006;367:1747-57. 
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Chidambaram JD, Melese M, Alemayehu W, et al. Mass antibiotic treatment and 
community protection in trachoma control programs. Clin Infect Dis 2004;39:e95-7. 
23. 
House JI, Ayele B, Porco TC, et al. Assessment of herd protection against 
trachoma due to repeated mass antibiotic distributions: a cluster-randomised trial. Lancet 
2009;373:1111-8. 
24. 
Goossens H, Ferech M, Vander Stichele R, Elseviers M. Outpatient antibiotic use 
in Europe and association with resistance: a cross-national database study. Lancet 
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Macrolide-resistant Streptococcus pneumoniae and use of antimicrobial agents. Clin 
Infect Dis 2001;33:483-8. 
26. 
Bronzwaer SL, Cars O, Buchholz U, et al. A European study on the relationship 
between antimicrobial use and antimicrobial resistance. Emerg Infect Dis 2002;8:278-82. 
27. 
Hotez PJ, Fenwick A, Savioli L, Molyneux DH. Rescuing the bottom billion 
through control of neglected tropical diseases. Lancet 2009;373:1570-5. 
28. 
Sommer A, Tarwotjo I, Djunaedi E, et al. Impact of vitamin A supplementation 
on childhood mortality. A randomised controlled community trial. Lancet 1986;1:1169-
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29. 
Rahmathullah L, Underwood BA, Thulasiraj RD, et al. Reduced mortality among 
children in southern India receiving a small weekly dose of vitamin A. N Engl J Med 
1990;323:929-35. 
30. 
Vijayaraghavan K, Radhaiah G, Prakasam BS, Sarma KV, Reddy V. Effect of 
massive dose vitamin A on morbidity and mortality in Indian children. Lancet 
1990;336:1342-5. 
31. 
West KP, Jr., Pokhrel RP, Katz J, et al. Efficacy of vitamin A in reducing 
preschool child mortality in Nepal. Lancet 1991;338:67-71. 
32. 
Vijayaraghavan K, Radhaiah G, Reddy V. Vitamin A supplementation and 
childhood mortality. Lancet 1992;340:1358-9. 
33. 
Vitamin A supplementation in northern Ghana: effects on clinic attendances, 
hospital admissions, and child mortality. Ghana VAST Study Team. Lancet 1993;342:7-
12. 
34. 
Daulaire NM, Starbuck ES, Houston RM, Church MS, Stukel TA, Pandey MR. 
Childhood mortality after a high dose of vitamin A in a high risk population. BMJ 
1992;304:207-10. 
35. 
Bryce J, Terreri N, Victora CG, et al. Countdown to 2015: tracking intervention 
coverage for child survival. Lancet 2006;368:1067-76. 
36. 
Jones G, Steketee RW, Black RE, Bhutta ZA, Morris SS. How many child deaths 
can we prevent this year? Lancet 2003;362:65-71. 
37. 
International Trachoma Initiative: Frequently Asked Questions. (Accessed 
November 3rd, 2011, at http://trachoma.org/iti-frequently-asked-questions.) 
38. 
Fenwick A, Molyneux D, Nantulya V. Achieving the Millennium Development 
Goals. Lancet 2005;365:1029-30. 
39. 
Verbal Autopsy Standards: Ascertaining and attributing causes of death. 2013. 
(Accessed March 6th, 2013, 2013, at 
http://www.who.int/healthinfo/statistics/verbalautopsystandards/en/.) 
40. 
Hsiang MS, Lin M, Dokomajilar C, et al. PCR-based pooling of dried blood spots 
for detection of malaria parasites: optimization and application to a cohort of Ugandan 
children. Journal of clinical microbiology 2010;48:3539-43. 
41. 
Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L. Detection of erythromycin-
resistant determinants by PCR. Antimicrobial agents and chemotherapy 1996;40:2562-6. 
42. 
Pai R, Gertz RE, Beall B. Sequential multiplex PCR approach for determining 
capsular serotypes of Streptococcus pneumoniae isolates. Journal of clinical 
microbiology 2006;44:124-31. 


 
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43. 
Tobias J, Vutukuru SR. Simple and rapid multiplex PCR for identification of the 
main human diarrheagenic Escherichia coli. Microbiol Res 2011. 
44. 
Norgen Biotek Corporation. Stool Nucleic Acid Collection and Transport Tubes 
Product Insert (PI45650-1). In; 2013. 
45. 
Steenkeste N, Incardona S, Chy S, et al. Towards high-throughput molecular 
detection of Plasmodium: new approaches and molecular markers. Malar J 2009;8:86. 
46. 
Qiagen. TopTaq PCR Handbook 2010. 
47. 
Qiagen. QIAquick Spin Handbook 2008. 
48. 
GE Healthcare. Nucleic Acid Sample Preparation for Downstream Analyses 
Principles and Methods Handbook.23-9. 
49. 
Norgen Biotek Corporation. Stool DNA Isolation Kit Product Insert (PI27600-3) 
In; 2009. 
50. 
Phuc Nguyen MC, Woerther PL, Bouvet M, Andremont A, Leclercq R, Canu A. 
Escherichia coli as reservoir for macrolide resistance genes. Emerg Infect Dis 
2009;15:1648-50. 
51. 
Nakhjavani FA, Emaneini M, Hosseini H, et al. Molecular analysis of typical and 
atypical enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea. 
Journal of medical microbiology 2013;62:191-5. 
52. 
Ramotar K, Waldhart B, Church D, Szumski R, Louie TJ. Direct detection of 
verotoxin-producing Escherichia coli in stool samples by PCR. Journal of clinical 
microbiology 1995;33:519-24. 
53. 
Wang RF, Cao WW, Cerniglia CE. PCR detection and quantitation of 
predominant anaerobic bacteria in human and animal fecal samples. Applied and 
environmental microbiology 1996;62:1242-7. 
54. 
Moller JK, Pedersen LN, Persson K. Comparison of the Abbott RealTime CT new 
formulation assay with two other commercial assays for detection of wild-type and new 
variant strains of Chlamydia trachomatis. Journal of clinical microbiology 2010;48:440-
3. 
55. 
Cheng A, Qian Q, Kirby JE. Evaluation of the Abbott RealTime CT/NG assay in 
comparison to the Roche Cobas Amplicor CT/NG assay. Journal of clinical microbiology 
2011;49:1294-300. 
56. 
Morre SA, van Dijk R, Meijer CJ, van den Brule AJ, Kjaer SK, Munk C. Pooling 
cervical swabs for detection of Chlamydia trachomatis by PCR: sensitivity, dilution, 
inhibition, and cost-saving aspects. Journal of clinical microbiology 2001;39:2375-6. 
57. 
Diamant J, Benis R, Schachter J, et al. Pooling of Chlamydia laboratory tests to 
determine the prevalence of ocular Chlamydia trachomatis infection. Ophthalmic 
Epidemiol 2001;8:109-17. 
58. 
Gebre T, Ayele B, Zerihun M, et al. Comparison of annual versus twice-yearly 
mass azithromycin treatment for hyperendemic trachoma in Ethiopia: a cluster-
randomised trial. Lancet 2012;379:143-51. 
59. 
Ellenberg SS FT, DeMets DL. Data monitoring committees in clinical trials: A 
practical perspective: John Wiley & Sons, LTD; 2003. 
 
 


 
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Appendix 
 
Niger 
Malawi 
Tanzania 
SAP 
Abbreviated Verbal Autopsy Questionnaire  
Infant Adverse Events survey 
Mobile App Manual  
Pfizer Investigator Initiated Research Serious Adverse Event Report Form and  
 
Completion Guide 
Resistance Breakpoints Table (to be added) 
Costing Forms (to be added) 
Data Management Plan (to be added) 


 
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MORDOR MOP Change Log 
Date 
Version Edit 
1 Apr 2015 
1.7 
Removed ear swabs from morbidity non-core activities 
1 Apr 2015 
1.7 
Removed symptom questionnaire from morbidity non-
core activities 
1 Apr 2015 
1.7 
Modification of section 6.1: Census Supervision.  
From: 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 will “lock” the census data 
collection for that community, and no changes can made be 
made to the census until the next census. 
To: 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. 
1 Apr 2015 
1.7 
Added option to use DNA/RNA shield media by Zymo 
for NP swab collection (Section 9) 
1 Apr 2015 
1.7 
Added option to use Norgen Stool Preservative for rectal 
swab collection (Section 9) 
1 Apr 2015 
1.7 
Added details for preparing DNA/RNA shield media 
(section 13.1.2) 
1 Apr 2015 
1.7 
Added 3.8kg minimum weight requirement for 
treatment (section 14) 
1 Apr 2015 
1.7 
Added: “Training will be in accordance with the 
Zithromax Program Manager’s Guide from the 
International Trachoma Initiative.” (Section 14.4) 
1 Apr 2015 
1.7 
Added: “An SAE report must be submitted for all deaths 
in the study – regardless of the time of treatment.” 
(Section 14.6.2) 
8 Sept 2015 
1.7 
Costing optional; clarification that dried blood spots are 
optional 
2 June 2016 
1.8 
Modification of Section 6.3: Verbal Autopsy, 
Questionnaire Administration. 
From: Our goal is to perform each verbal autopsy within 2 
months of death. 
To: Our goal is to perform each verbal autopsy within 2 
months of identification of death.   
2 June 2016 
1.8 
Modification of Section 4.3: Study Schedule table. 
From:  Fall, Winter, Spring, and Summer. 
To: Month specific dates i.e. Jan 15-July 15 for each 


 
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77 | P a g e  
 
phase.    
2 June 2016 
1.8 
Modification of Section 4.3: Study Schedule table. 
From: Malaria Collection occurring between MORDOR 
18 and 24 
To: Malaria Collection as part of MORDOR 6  
2 June 2016 
1.8 
Added in Section 4.3: Study Schedule table:  
Addition of *: +/- 1 month   
2 June 2016 
1.8 
Modification of Section 4.3: Study Schedule table legend 
narrative. 
From: …anthropometry from all children aged… 
To: …anthropometry from a cohort of children aged… 
2 June 2016 
1.8 
Added: “TL will also forward SAE to DSMC if meets 
criteria of being possibly related to study drug.” (Section 
14.6.2) 
2 June 2016 
1.8 
Added: “paper” to Supplies 
Added: “with pieces of paper in between each sample.” 
to Note 
(Section 8.2: Set-up drying area for TropBio bloodspots) 
2 June 2016 
1.8 
Added Section 18.3 Data Monitoring and Cleaning 
27 June 2018 
1.9 
Clarified how deaths as part of the primary outcome will 
be reported to Pfizer. 
24 October 
2019 
1.10 
Updated section 17 to reflect the MORDOR Contingency 
study Years 4-5 DSMC  
 

SAP_001.pdf: 
1 
 Mortality Reduction  after Oral Azithromycin 
Contingency Study, Niger, Year 3 and after 
NCT 03338244 
November 2020 
Statistical Analysis Plan 
UCSF Francis I. Proctor Foundation 
The Carter Center 
Niger Ministry of Health 
Johns Hopkins University Dana Center and Bloomberg School of Public Health 
Tanzania National Institute for Medical Research and Muhumbili Medical 
Center 
London School of Hygiene and Tropical Medicine 
University of Malawi, Blantyre College of Medicine 
Malawi Ministry of Health 


2 
 


3 
Introduction 
This document (Statistical Analysis Plan, SAP) describes the planned analysis and reporting for 
the clinical trial, Mortality Reduction after Oral Azithromycin, Contingency Study, Years 3-4, 
Niger. It includes specifications for the statistical analyses and tables to be prepared for the 
interim and final Clinical Study Report. This study is a Phase IV clinical trial to compare 
methods to reduce childhood mortality using mass administration of azithromycin (Pfizer, CAS 
83905-01-5) compared to placebo. The content of this Statistical Analysis Plan meets the 
requirements stated by the US Food and Drug Administration and conforms to the American 
Statistical Association’s Ethical Guidelines. 
The following documents were reviewed in preparation of this Statistical Analysis Plan:  
• Mortality Reduction after Oral Azithromycin, Protocol  
• Mortality Reduction after Oral Azithromycin, Statistical Analysis Plan 
• Mortality Reduction after Oral Azithromycin, Manual of Operations  
• ICH Guidance on Statistical Principles for Clinical Trials  
The planned analyses described in this SAP will be included in future manuscripts. Exploratory 
analyses not necessarily identified in this Statistical Analysis Plan may be performed to support 
the analysis. Unplanned analyses not delineated in this Statistical Analysis 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. 
 


4 
MORDOR Stage 2 Contingency Study 
 
1 Introduction 
 
Should the primary analysis of azithromycin to prevent mortality reveal evidence of a benefit, we 
propose to proceed with two additional studies in Stage 2.  
 
2 Design 
 
Contingency study A. All communities in Niger will receive treatment at the conclusion of 
MORDOR Stage 1 (original study).  Specifically, azithromycin communities will continue with 
azithromycin, and placebo communities will receive azithromycin at the 24 month visit (census 
number 5). Follow-up will occur at month 30 and month 36, providing one additional year of 
data.  
 
Contingency study B. At the 36 month visit, rerandomization will occur, and half of communities 
will begin to receive placebo.  
 
3 Analysis 
 
The primary outcome is mortality, analyzed in the same way as in MORDOR Stage I, i.e., 
present on an initial census, and absent on the next follow-up census due to death. Since the study 
has been extended, the same analytic template will be used for each visit, e.g. at 48, 54, and 60 
months. Each visit that is conducted will be reported. 
 
Study A: 
1. Sample size considerations. 
a. Assuming communities of size 668 with approximately 17% in the target age 
range, an effect size of 20%, 10% loss to follow-up per year, 2% mortality per 
year, and a CV of 0.51, 312 communities per arm provides approximately 87% 
power (see MORDOR Phase I SAP for details). 
b. Updating based on data from Phase I (Niger), we find the following. 
Assuming the observed CV of 0.34, a death rate of 2% per year results in  
approximately 80% power to detect a 17% effect. A death rate of 2.5% per 
year (and a CV of 0.34) result in approximately 80% power to detect a 15.5% 
effect size. 
2. Primary prespecified analysis: We propose to compare the former placebo arms with 
the former azithromycin arms, using negative binomial regression. Significance 
testing is two-sided with an alpha of 0.05, and will be based on permutation testing. 
(This analysis is identical in every way to the country subgroup analyses conducted in 
Stage 1). This is restricted to the period from 24 months to 36 months, covering two 
six-month study intervals. 


5 
3. We propose, as a prespecified secondary analysis, a longitudinal comparison of the 
original placebo communities. We propose to contrast the first four study intervals 
during which these communities received placebo, to the last two study intervals 
during which these communities received azithromycin. Analysis will be conducted 
using negative binomial regression, with a permutation P-value reported (two-sided, 
alpha=0.05). The community-randomized nature of the trial is thus taken into account. 
4. Additional supplementary analyses will be distinguished from the two prespecified 
analyses above. In particular, the following will be reported: 
a. Analysis of the seasonality and timing of deaths: time of death, time of 
treatment for those who died, and time since treatment for all deaths. We will 
conduct comparisons of time since treatment conditional on death based on the 
Cramer-von Mises test (exactly as in Stage 1), additionally comparing across 
treatment arms and longitudinally. Comparisons of equality of circular means 
will be used to assess seasonality of death and of treatment, comparing 
between randomization arms and longitudinally. We will, in particular, focus 
special attention and reporting to a comparison of phases 1-4 to 5-6 within the 
placebo arm. 
b. Analysis will be reported according to the same age classes used in the 
original study (Keenan et al, NEJM, 2018), i.e. 1-5, 6-11, 12-23, 24-59 
months. 
c. We will examine the difference between phase 5 and 6 in the same way. 
d. We will report longitudinal analysis of mortality changes in the original 
azithromycin arm, using a linear predictor of study interval (1—6).  
e. We will compare estimated treatment effect at a regional level using placebo 
mortality rates as a predictor. 
 
Study B (years two and three of mortality contingency study) 
1. Sample size considerations are the same as in Study A (year 1).  
a. Assuming communities of size 668 with approximately 17% in the target age 
range, an effect size of 20%, 10% loss to follow-up per year, 2% mortality per 
year, and a CV of 0.51, 312 communities per arm provides approximately 87% 
power (see MORDOR Phase I SAP for details). 
b. Updating based on data from Phase I (Niger), we find the following. Assuming the 
observed CV of 0.34, a death rate of 2% per year results in  approximately 80% 
power to detect a 17% effect. A death rate of 2.5% per year (and a CV of 0.34) 
result in approximately 80% power to detect a 15.5% effect size. 
2. The primary prespecified analysis will contrast the rerandomized placebo arm with the 
rerandomized azithromycin arm. This will be conducted with negative binomial 
regression, using a two-sided alpha of 0.05 and a permutation test by randomization arm.  
3. Supplementary analyses will control for prior history (original randomization arm). 
4. Longitudinal analysis for communities who always received azithromycin will be 
reported. 
5. We will compare all four groups for the last year of data (two histories by two final 
treatments). 


6 
6. Additional analyses by the same age groups as before and by phase will be reported. 
7. Models of timing, seasonality, and geography will be reported as secondary analysis. 
 
Morbidity Study. Analysis of morbidity communities will follow the exact template as Stage I. 
Primary prespecified analyses will be two-sided, at an alpha of 0.05. However, the morbidity 
communities are still receiving placebo. Note that we will report the full follow-up at 48 months 
separately from the Stage 1 primary analyses. We will also look for secular effects longitudinally 
over the four years of the full study, as well as phases 5-8 separately. 
1. Anthropometry: we will examine height and weight longitudinally, and WFH Z score 
secondarily. Please see the Stage 1 SAP for details. 
2. Other analyses will include drug resistance and malaria, as in Stage 1. 
3. Note that we do not do nares or conjunctiva swabs, conjunctival photos, or any swabs 
on 7-12 year olds in the contingency study. 
 
Statistical considerations 
1. Communities which drop out of the study will be omitted from analysis for all study 
period for which we have no data for that community. If a community rejoins at a 
particular phase, it will be included as before. 
2. Evidence of poor model fit to the negative binomial regression will justify reporting 
additional models. 
3. Permutation tests will be conducted with 10000 replications. If the critical value 0.05 lies 
within the Monte Carlo confidence interval for the P-value, we will replicate with 
10,000,000 replications, and this will be reported. 
 
Resistance comparison. 
160 total communities from the mortality contingency study B (above) will be randomized to the 
4 arms (blocked on baseline assignment, but not on rerandomization), and specimens collected 
from each of 36 children randomly selected from the census. The children will be selected from 
the age range 1-59 months (inclusive), at the 54 month census. The following outcomes are to be 
assessed: 
 
Rectal Swab samples: 
1) Outcome: Normalized read number for macrolide resistance determinants from DNA 
sequencing. These are to be physically pooled by community prior to sequencing; 
individual specimens will not be analyzed separately, and individual-level analysis will 
not be possible. 
Analysis: We propose to use pairwise Wilcoxon rank-sum tests between each of the four 
treatment history groups, accounting for multiple comparisons as described  [1-3]. All 
randomized communities will be included in the permutation testing. 
This is the primary analysis for this study. All other analyses for the rectal swab 
collection (below) are secondary. 
As a methodological sensitivity analysis, the Kruskal-Wallis (nonparametric one-way 
ANOVA), comparing all four arms. Such sensitivity analyses will be sharply 
distinguished from the primary prespecified analysis. 


7 
Mean fold difference between arms will be reported or plotted, as well. 
2) Normalized read number of Campylobacter at the genus and species levels, derived from 
DNA sequencing. We  will systematically compare the relative abundances of individual 
microbial taxa between groups at the species level by negative binomial Wald test using 
the Benjamini–Hochberg procedure for controlling FDR [4]. Differentially abundant taxa 
with a false discovery rate < 0.05 and a twofold change will be considered as notable. 
Sensitivity test will be perform by first using the two-sided Kruskal–Wallis test at a 
predefined alpha of 0.05. Significantly different vectors resulting from the comparison of 
relative abundances between any two groups will be used as input for linear discriminant 
analysis (LDA), which produces an effect size [5]. The LDA will be set at 2 for all 
analyses. 
3) Beta lactam resistance determinants will be analyzed, as in 1) above. 
4) Other analyses will be conducted using the same pooling method, but will feature the use 
of Benjamini-Hochberg FDR procedures. The FDR is prespecified at 0.05. 
a. Non-macrolide resistance determinants other than beta-lactam 
b. Occurrence of different viruses or bacteria (we exclude eukaryotic organisms). Of 
particular importance are Chlamydia spp., malaria, and tuberculosis. 
5) As in 1) above, but with the following outcome for each community: bacterial species 
normalized read counts. Statistically, we propose PERMANOVA comparison of all 4 
arms, based on the L2 norm. As a sensitivity analysis, we will compare using the L1 norm. 
6) As in 4), but with species diversity as the primary outcome. We propose use of the L2 
norm for the primary analysis, and then, as a sensitivity analysis, we will compare using 
the L1 norm, both expressed as effective number. The P-values will be derived from 
permutation. 
 
Sample size. We chose N=160 total comunities, which provides approximately 80% power to 
detect a 3.5-fold difference in any pairwise comparison (assuming P=0.01, and an approximate 
SD of the base two logarithm of the read count (plus 1) of 2.25). The SD of the the log read count 
was derived from the placebo arm of the MORDOR 36 month data. The effect size (3.5 fold) is 
approximately half of what was observed in the MORDOR 36 month data. 
 
NP Swab samples: 
1) Analysis will compare macrolide resistance at the community level, using the Westfall-
Young method [1] as above. This is the primary analysis for the NP swab study. 
2) Exploratory analysis comparing the occurrence of different organisms at the genus and 
species levels. We prespecify an FDR of 0.05.  
 
References 
 
[1] Westfall PH, Troendle JF. Multiple testing with minimal assumptions. Biometrical Journal 
50(5): 745–755, 2008. 


8 
[2] Ge Y, Dudoit S, Speed TP.  Resampling-based multiple testing for microarray data analysis. 
Test 12: 1–77, 2003. 
[3] Westfall PH, Young SS. Resampling-Based Multiple Testing: Examples and Methods for p-
Value Adjustment. Wiley, New York, 1993. 
[4] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-
seq data with DESeq2. Genome biology 2014, 15:550. 
[5] Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C: 
Metagenomic biomarker discovery and explanation. Genome biology 2011, 12:R60.