LAKANA , a Cluster-randomized, Double-blinded, Parallel Group, Controlled Trial, Testing the Effects of Mass Drug Administration of Azithromycin on Mortality and Other Outcomes Among 1-11 Month Old Infants in Rural Mali.
Phase: PHASE3
Conditions: Mortality
Sponsor: Tampere University

Protocol version 7.0: 13. November. 2024 
LAKANA trial 
 
 
A research plan for a cluster-randomized, double-blinded, parallel group, 
controlled trial, testing the effects of mass-drug administration of azithromycin on 
mortality and other outcomes among 1–11-month-old infants in rural Mali 
 
 
Principal and co-principal investigators (in alphabetical order) 
Per Ashorn, MD, PhD 1 
Ulla Ashorn, PhD 1 
Yin Bun Cheung, PhD 1,2 
Camilla Ducker, MBBS, MSc 3 
Nigel Klein, MBBS, PhD 4 
Samba O Sow, MD, MSc, FASTMH 5 
 
 
Participating Academic Institutions 
 
1Center for Child Health Research, Tampere University, Faculty of Medicine and 
Health Technology, Tampere, Finland 
2Centre for Quantitative Medicine, Duke-NUS Medical School, Singapore 
3Tro Da Ltd, UK 
4University College London, UK 
5CVD-Mali, Bamako, Mali 
 
 
Contact information: Per Ashorn, MD, PhD, Center for Child Health Research, 
Tampere University, Faculty of Medicine and Health Technology, Tampere 
Finland 
Tel. +358 40 7280 345, E-mail: per.ashorn@tuni.fi  

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Protocol version 7.0: 13. November. 2024 
 
Table of contents 
 
1. PROTOCOL VERSION HISTORY ......................................................................... 4 
2. EXECUTIVE SUMMARY ........................................................................................ 9 
2.01. ADMINISTRATIVE INFORMATION ........................................................................... 9 
2.02. KEY DETAILS OF THE TRIAL ................................................................................ 10 
3. BACKGROUND ....................................................................................................... 12 
3.01. HEALTH PROBLEM TO BE ADDRESSED ................................................................. 12 
3.02. STUDY OBJECTIVE ............................................................................................... 12 
3.03. BACKGROUND TO AND JUSTIFICATION FOR THE STUDY ....................................... 12 
3.04. POTENTIAL PUBLIC HEALTH EFFECTS .................................................................. 13 
3.05. DISTRIBUTION OF THE RESULTS .......................................................................... 14 
4. THE STUDY ............................................................................................................. 14 
4.01. OBJECTIVES OF THE PROPOSED RESEARCH .......................................................... 14 
4.02. DETAILED STUDY QUESTIONS ............................................................................. 15 
4.03. STUDY SITE AND TARGET POPULATION ............................................................... 17 
4.04. STUDY DESIGN .................................................................................................... 17 
4.05. ENROLLMENT CRITERIA FOR PARTICIPATING CLUSTERS AND PARTICIPANTS ....... 18 
4.06. TRIAL INTERVENTIONS AND CONTROL ................................................................ 19 
4.07. OUTCOME VARIABLES ......................................................................................... 19 
4.08. DATA COLLECTION SITES FOR VARIOUS OUTCOMES ............................................ 22 
4.09. SCHEDULE AND PROCEDURES FOR PARTICIPANT ENROLLMENT AND FOLLOW-UP 23 
4.10. EXPECTED RECRUITMENT AND LOSS-TO-FOLLOW-UP RATES ............................... 25 
4.11. PURCHASE, FORMULATION, STORAGE, AND DISTRIBUTION OF STUDY DRUGS ...... 26 
4.12. DATA COLLECTION ON INFANT AND CHILD MORTALITY ...................................... 27 
4.13. ADAPTIVE DESIGN: POSSIBLE CHANGES AFTER AN INTERIM ANALYSIS ................ 27 
4.14. DATA COLLECTION ON INFANT MORBIDITY ......................................................... 29 
4.15. DATA COLLECTION ON INFANT GROWTH AND NUTRITIONAL STATUS .................. 29 
4.16. DATA COLLECTION ON ANTIMICROBIAL RESISTANCE .......................................... 30 
4.17. DATA COLLECTION ON MECHANISMS OF ACTION FOR AZITHROMYCIN ................ 36 
4.18. STAKEHOLDER PERSPECTIVES ON FEASIBILITY, INCLUDING ECONOMIC ANALYSES41 
4.19. STRATEGIE AVANCÉE AND FIELD WORKERS’ ASSESSMENT OF HARD-TO-REACH AREAS45 
4.20. DATA COLLECTION ON TRIAL SAFETY ................................................................. 46 
4.21. GROUP ALLOCATION AND TRIAL CODE MANAGAMENT ........................................ 46 
4.22. METHODS FOR PROTECTING AGAINST OTHER SOURCES OF BIAS .......................... 47 
4.23. WITHDRAWAL FROM PARTICIPATION AFTER ENROLLMENT ................................. 48 
4.24. OTHER TREATMENTS TO TRIAL PARTICIPANTS .................................................... 48 
4.25. CO-ENROLLMENT GUIDELINES ............................................................................ 48 
4.26. STORAGE OF BIOLOGICAL SAMPLES .................................................................... 48 
4.27. DATA RECORDING AND MANAGEMENT ............................................................... 49 
4.28. PILOT STUDY ....................................................................................................... 50 
4.29. QUALITY ASSURANCE AND CONTROL .................................................................. 50 
4.30. ORIGINALLY PLANNED TIME SCHEDULE .............................................................. 50 

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Protocol version 7.0: 13. November. 2024 
 
5. STATISTICAL CONSIDERATIONS AND DATA ANALYSIS ......................... 52 
5.01. SPECIFIC HYPOTHESES ........................................................................................ 52 
5.02. ANALYTICAL APPROACH ..................................................................................... 53 
5.03. FREQUENCY OF ANALYSES .................................................................................. 55 
5.04. SAMPLE SIZE AND ITS JUSTIFICATION .................................................................. 56 
5.05. SUBGROUP ANALYSES ......................................................................................... 57 
6. ETHICAL CONSIDERATIONS ............................................................................ 58 
6.01. GENERAL PRINCIPLES ......................................................................................... 58 
6.02. INFORMED CONSENT ........................................................................................... 58 
6.03. POSSIBLE RISKS AND BENEFITS TO STUDY PARTICIPANTS .................................... 59 
6.04. AMOUNT OF COLLECTED BLOOD AND OTHER BIOSPECIMENS .............................. 60 
6.05. INSURANCE COVERAGE ....................................................................................... 61 
6.06. COMPENSATION TO STUDY PARTICIPANTS........................................................... 61 
6.07. PAYMENTS TO THE TRIAL ORGANISERS ............................................................... 61 
6.08. DATA SAFETY AND MONITORING BOARD (DSMB) .............................................. 62 
6.09. STOPPING RULES FOR THE STUDY ........................................................................ 62 
7. PERSONNEL, RESOURCES, AND STUDY MANAGEMENT ......................... 63 
7.01. RESEARCH INSTITUTIONS .................................................................................... 63 
7.02. PRINCIPAL INVESTIGATORS ................................................................................. 63 
7.03. STUDY STATISTICIANS ........................................................................................ 65 
7.04. OTHER MAIN SCIENTISTS AND THEIR RESPONSIBILITIES ...................................... 65 
7.05. OTHER EMPLOYED STAFF .................................................................................... 65 
7.06. ARRANGEMENTS FOR DAY-TO-DAY MANAGEMENT OF THE STUDY ...................... 66 
7.07. COMMUNICATION PLAN ...................................................................................... 67 
7.08. PROJECT STEERING GROUP ................................................................................. 67 
7.09. NATIONAL AND INTERNATIONAL ADVISORY GROUPS .......................................... 68 
8. TRAINING PROVIDED ......................................................................................... 68 
9. POSSIBLE CONSTRAINTS ................................................................................... 68 
10. FUNDING ............................................................................................................... 69 
11. REFERENCES ....................................................................................................... 70 
12. APPENDICES ......................................................................................................... 77 
APPENDIX 1A: INFORMATION ABOUT THE TRIAL TO VILLAGE LEADERSHIP .................. 77 
APPENDIX 1B: VERBAL CONSENT FORM FOR VILLAGE PARTICIPATION ....................... 83 
APPENDIX 2A: INFORMATION ABOUT THE TRIAL TO A POTENTIAL PARTICIPANT .......... 84 
APPENDIX 2B: VERBAL HOUSEHOLD CONSENT FORM FOR TRIAL PARTICIPATION ....... 90 
APPENDIX 3: CONSENT TO PROVIDE STUDY MEDICINE TO AN INDIVIDUAL INFANT ...... 91 
APPENDIX 4A: INFORMATION ON SECONDARY OUTCOME DATA COLLECTION .............. 93 
APPENDIX 4B: CONSENT FORM FOR PARTICIPATION IN LAKANA SUB-STUDIES ........ 98 
APPENDIX 5, REFERRAL LETTER FOR ASYMPTOMATIC CHILDREN WITH ABNORMAL LABORATORY VALUES 
OBSERVED DURING A VILLAGE VISIT .......................................................................... 102 
 

Protocol version 7.0: 13. November. 2024 
1. Protocol version history 
1 
 
2 
AMENDME
NT NO. 
PROTOCOL 
VERSION # 
DATE ISSUED 
AUTHOR/S 
OF 
CHANGES 
DETAILS OF CHANGES MADE 
SUBMITTED 
TO IRB, 
MALI 
(DATE) 
APPROVED 
AT IRB , 
MALI 
(DATE/NO) 
SUBMITTED 
TO IRB , 
TAU (DATE) 
APPROVED 
AT IRB, 
TAU 
(DATE/NO) 
 
1.0 
27.11.2019 
Ashorn 
First complete protocol draft. Submitted for IRB review but 
withdrawn by the research team for further edits 
29.11.2019 
Withdrawn 
N / A 
N / A 
1 
2.0 
17.12.2019 
Ashorn 
Expanded the trial area: Earlier only Kayes region, now parts of 
Kayes, Kita, and Koulikoro regions. 
Removed the bridging trial aftern interim analysis 
Cut out the last 6 calendar months of follow-up in the trial. Half of 
the clusters now followed up for 8 MDA rounds, 30% for 7 MDA 
rounds and 20% for 6 MDA rounds 
Increasing sample size from 650 to 750 mortality analysis clusters 
Changed the ratio of control vs azi-biannual vs azi quarterly clusters 
from 1 : 1 : 1 to 3 : 4 : 2 
Reduced the number of interim analyses from 2 to 1 – to be done at 
approximately 60% of the data accrual 
Changed the timing of AMR and other biological sample collection 
slightly 
Added a second field laboratory to Koulikoro region 
Expanded the area of a possible MDA roll-out area to 3-5 regions, 
including the entire Kayes, Kita and Koulikoro, and possibly parts 
of Bougouni and Segou. 
17.12.2019 
27.12.2019 
13.01.2020 
04.02.2020 
2 
3.0 
08.03.2022 
Luoma, 
Adubra, 
Ashorn 
Increased sample size from 830 to 1150 clusters (or 35,650 infants 
treated on MDA1, whichever comes first), because of lower-than-
expected numbers of infants / cluster and baseline mortality. Edited 
assumptions behind the sample size selection. 
Changed the plan of primary analysis, to include mortality data also 
from the secondary outcome sample. Clarified the process of code 
breaking before primary analysis 
15.3.2022 
28.3.2022 
 
 

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AMENDME
NT NO. 
PROTOCOL 
VERSION # 
DATE ISSUED 
AUTHOR/S 
OF 
CHANGES 
DETAILS OF CHANGES MADE 
SUBMITTED 
TO IRB, 
MALI 
(DATE) 
APPROVED 
AT IRB , 
MALI 
(DATE/NO) 
SUBMITTED 
TO IRB , 
TAU (DATE) 
APPROVED 
AT IRB, 
TAU 
(DATE/NO) 
Extended the duration of cluster enrolment from 52 to 78 weeks 
Removed the inclusion of a tertiary AMR sample 
Changed the timing for AMR analyses to include samples at baseline, 
and at 12, 24, ad 36 months after baseline 
Clarified the AMR analyses to include resistance to WHO “Access” 
group antibiotics among azithromycin-resistant bacterial isolates 
Edited laboratory tests to be done in the mechanistic study: replaced 
fecal calprotectin measurement with three other fecal biomarkers: 
neopterin, myeloperoxidase, and alpha 1 antitrypsin 
Provided more detail on the age of infants eligible for the 
intervention (aged expressed in days, not only months) 
Provided further details on possible outcome scenarios for the 
planned interim analysis 
Change establishment of a second field site for AMR testing in 
Koulikoro as optional. 
Edited participant information document with the updated 
information on sample size and age of eligible infants 
Provided detailed instructions regarding study drug administration 
and referral of children participating in mechanistic substudy for 
whom blood samples are collected and Hb and CRP levels are 
measured on site. Added a document that will be used to advice 
CSCOM personnel on participant management (Appendix 5). 
3 
4.0 
27.06.2022 
Luoma, 
Adubra, 
Ashorn 
Added a tertiary sample for collection of AMR and mechanistic study 
samples in a subset of villages closer to Bamako, i.e., in the study 
regions of Koulikoro and Kita. 
Replaced Norgen Stool Nucleic Acid kit by DESS media 
(DMSO/EDTA/saturated sodium chloride) for the preservation of 
second rectal swab collected at AMR study visits.  
Specified that AMR and mechanistic study samples may be directly 
transported to CVD-Mali Bamako laboratory for processing. 
 
 
 
 

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AMENDME
NT NO. 
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VERSION # 
DATE ISSUED 
AUTHOR/S 
OF 
CHANGES 
DETAILS OF CHANGES MADE 
SUBMITTED 
TO IRB, 
MALI 
(DATE) 
APPROVED 
AT IRB , 
MALI 
(DATE/NO) 
SUBMITTED 
TO IRB , 
TAU (DATE) 
APPROVED 
AT IRB, 
TAU 
(DATE/NO) 
Changed the timing for mechanistic study data and sample collection 
to MDA 4 for 4–11-month-old infants, MDA 6-8 and visit 9 for 6-
8- and 12-14-months old infants and children. 
Edited one hypothesis for the mechanistic study extending the 
assessment of community-level prevalence of malaria parasitaemia 
at the end of the intervention (9th visit) to 12-14 months old 
children.  
Edited instructions regarding referral of children who have abnormal 
Hb or CRP values in the mechanistic sub-study. Edited the referral 
document (Appendix 5). 
Specified that anthropometric measurements for growth study, and 
samples collection for mechanistic study will be conducted at the 
time of the study visits, instead of two weeks later, and possibly in 
a central location in village.  
Removed head circumference from growth study outcomes.  
Changed the target age for morbidity data collection to 4-14 months.   
Provided further details on how the feasibility study will be done. 
Added an economic analysis (chapter 4.18). 
Clarified, based on trial adaptive design, the interim analysis 
outcomes that will lead trial conduct to be altered. 
Specified that a 1:1 allocation ratio will be used to re-randomize 
previous placebo-clusters in the two AZI groups if after interim 
analysis the placebo arm is dropped.  
Clarified that random allocation of letter codes to treatment was 
conducted by the external partner, RTI International, who then 
provided the code to Pfizer. 
Clarified that closed-testing procedure will be used to adjust 
statistical analyses for multiple comparisons. 
Clarified that analytical method for mechanism study will account for 
clustering. 
 

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Protocol version 7.0: 13. November. 2024 
 
AMENDME
NT NO. 
PROTOCOL 
VERSION # 
DATE ISSUED 
AUTHOR/S 
OF 
CHANGES 
DETAILS OF CHANGES MADE 
SUBMITTED 
TO IRB, 
MALI 
(DATE) 
APPROVED 
AT IRB , 
MALI 
(DATE/NO) 
SUBMITTED 
TO IRB , 
TAU (DATE) 
APPROVED 
AT IRB, 
TAU 
(DATE/NO) 
4 
5.0 
23.03.2023 
Luoma, 
Adubra, 
Ashorn 
Specified that mechanistic study in tertiary sample will be conducted 
during rainy season (Peak malaria transmission). 
Specified that Growth study may be conducted in tertiary sample if 
planned sample size for the study is not achieved with secondary 
sample.  
Specified “Parasite density” as an outcome of mechanistic study to 
clarify the quantitative aspect of parasitaemia measures besides 
malaria prevalence.  
Provided clarification on exploratory analysis and effect modifiers to 
be tested.  
Edited one hypothesis for the mechanistic study extending the 
assessment of community-level prevalence of malaria parasitaemia 
at the end of the intervention (9th visit) to 1-59 months old 
children. And, provided further details on sample size and 
sampling methods, as well as compensation.  
Edited the Information about Participation in the AMR and other sub-
studies to include Kati as one of the CSComs included in the sub 
studies (tertiary sample).  
 
 
 
 
 
5 
6.0 
15.12.2023 
Alber, 
Fan, 
Klein 
 
Expanded the age group of children being sampled for AMR analysis 
at visit 11 to also include 15-26 months old children. Expanding the 
age group at visit 11 will allow to assess whether carriage of 
antibiotic resistant bacteria persists in individual children who 
received 1-4 doses of azithromycin MDA one year prior to sampling 
and who live in a village that have stopped MDA intervention.  
Additionally, we will sample 24-35 months old children at visit 9 in 
the Kati region (tertiary AMR sample). This will determine whether 
carriage of antibiotic resistant bacteria persist in children who 
received 1-4 doses of azithromycin MDA one year prior to sampling 
but live in a village where MDA intervention has continued. 
 
 
 
 

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Protocol version 7.0: 13. November. 2024 
 
AMENDME
NT NO. 
PROTOCOL 
VERSION # 
DATE ISSUED 
AUTHOR/S 
OF 
CHANGES 
DETAILS OF CHANGES MADE 
SUBMITTED 
TO IRB, 
MALI 
(DATE) 
APPROVED 
AT IRB , 
MALI 
(DATE/NO) 
SUBMITTED 
TO IRB , 
TAU (DATE) 
APPROVED 
AT IRB, 
TAU 
(DATE/NO) 
6 
7 
13.11.2024 
Luoma, 
Ashorn 
Added “hard-to-reach” village status and Strategie Avancée as one of 
the potential effect modifiers in sections 4 and 5. 
Included a survey among the field workers as a part of the feasibility 
aspect. 
Amended a study question regarding hardness-to-reach and the 
inclusion of village to Strategie Avancée being a predictor of high 
mortality (section 4.02.) 
Amended the aspect of field worker assessment to section 4.18. 
 
 
 
 
1 

Protocol version 7.0: 13. November. 2024 
2. Executive summary 
 
2.01. Administrative information 
 
Trial name: 
LAKANA. A cluster-randomized, double-blinded, parallel 
group, controlled trial, testing the effects of mass-drug 
administration of azithromycin on mortality and other 
outcomes among 1–11-month-old infants in rural Mali 
 
Sponsor: 
Tampere University, FIN-33014 Tampere, Finland 
 
Funding: 
The Bill & Melinda Gates Foundation 
 
Monitor: 
Likak Research Ltd, Senegal 
 
Data Safety and 
Dr. Robert Black, JHU, chair (Global child health) 
Monitoring Board: 
Dr. Julia Bielicki, UKBB, Basel, Switzerland (AMR) 
 
Dr. Alassane Dicko, USTTB, Bamako, Mali (Epidemiology) 
 
Dr. Queen Dube, COM, Malawi (Child health policy) 
 
Dr. Paul Milligan, LSHTM, London, UK (Biostatistics) 
 
Principal Investigator 
Dr. Per Ashorn, MD, PhD 
 
Co- Principal Investigators: Dr. Samba Sow, MD, MSc, FASTMH 
 
Dr. Ulla Ashorn, PhD 
 
Dr. Nigel Klein, MBBS, PhD 
 
Dr. Camilla Ducker, MBBS, MSc 
 
Dr Yin Bun Cheung, PhD 
 
Participating institutions:  Tampere University, Finland 
 
CVD-Mali, Bamako, Mali 
 
University College London, UK 
 
Tro Da Ltd, UK 
 
Centre for Quantitative Medicine, Duke-NUS Medical 
School, Singapore 
 
Trial site: 
Kayes, Kita, and Koulikoro regions, Mali 
 
Laboratory analyses: 
All participating institutions 
 
Contact details: 
Dr. Per Ashorn, MD, PhD 
 
Professor of Paediatrics, Center for Child Health Research, 
Faculty of Medicine and Health Technology 
 
Tampere University, FIN-33014 Tampere, Finland 
 
Tel: +358 40 7280 354, Email: per.ashorn@tuni.fi 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
2.02. Key details of the trial 
 
Background:  
There is recent evidence that mass-drug administration (MDA) of 
azithromycin to apparently healthy young children, reduces their 
mortality in some, but not in all low-income African settings.  
 
Main objective:  
To determine the impact of quarterly (given every 3 months) or bi-
annual (given twice / year, between January and June) azithromycin 
MDA to 1–11-month (29-364 days) old infants  on their mortality 
and other health outcomes, when provided in a context of a seasonal 
malaria chemoprevention (SMC) program. 
 
Trial approach: 
A cluster-randomized, placebo-controlled, double-blinded, parallel-
group, three-arm clinical trial with adaptive design, in rural and peri-
urban villages in the Kayes, Kita, and Koulikoro regions of Mali, 
Sub-Saharan Africa. 
 
Main methods: 
Selected and consenting villages will be randomly allocated into 
three groups: Control, Azi-quarterly, and Azi-biannual. Households 
in the participating villages will be visited at quarterly intervals by 
study staff. At each visit, there will be a census of all household 
members as well as recording of births and child deaths since the last 
visit. All 1-11 months old infants (age 29-364 days), for whom there 
is a consent from a guardian for study drug provision, will be 
weighed and given a single dose of study drug. Infants in control 
group will receive placebo at each visit, infants in Azi-bianual group 
will receive placebo at visits between July and December and 20 mg 
/ kg azithromycin at visits between January and June, and infants in 
the Azi-quarterly group will receive 20 mg / kg azithromycin at each 
visit. The village visit will be repeated every 3 months for 8 times 
for study drug administration and one more time for census and vital 
statistics recording. SMC with amodiaquine and sulfadoxine-
pyrimethamine will be offered through the national health service to 
all 3–59-month-old children on a monthly basis between July and 
October in each year. 
 
Primary outcome Mortality (deaths / 1000 years at risk) among children who were 1-
11 month (29-364 days) old at mass azithromycin administration. 
 
Other outcomes 
Episodes of ARI, malaria, and diarrhea 
Infant and young child growth 
Prevalence of antimicrobial resistance towards azithromycin 
Malaria parasitemia, inflammation, and immune development 
Feasibility of azithromycin MDA implementation including 
economic analysis 
Incidence of serious adverse events (SAE) and adverse events (AE) 
Mortality among children, who were 12–59-month-old at the MDA 
 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
Adaptive design: 
One interim analysis will be completed when approximately 60% of 
the 3-month follow-up intervals have been completed. If there is a 
statistically significant mortality difference between the quarterly 
azithromycin group and the placebo group as well as between the 
biannual azithromycin group and the placebo group but not between 
the two azithromycin groups, the placebo arm will be discontinued, 
and trial completed with two arms only. If there is also a statistically 
significant difference also between the two azithromycin groups, the 
trial will be stopped, and the team will offer to work with Malian 
Ministry of Health to provide azithromycin MDA in the trial site and 
elsewhere in the regions of Mali. 
 
Sample size: 
Approximately 1150 clusters (villages). At each MDA round, each 
cluster is expected to contain on average 31 eligible infants who will 
provide data to the study. 
 
Stored samples: 
Some vials of frozen plasma, white blood cells, urine and stools and 
dried blood spots from a sub-group will be stored for later analyses. 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
3. Background 
 
 
3.01. Health problem to be addressed 
 
Infant and child mortality in Mali and elsewhere in sub-Saharan Africa.  
 
According to the most recent estimates, 3.0 million infants and children between 1-59 
months die each year, mostly from infectious diseases (WHO, 2018). Approximately 1.2 
million of these deaths are estimated to take place in Western and Central Africa, 0.7 
million in Eastern and Southern Africa, 0.7 million in in South Asia, 0.3 million in East 
Asia and Pacific, and 0.1 million elsewhere (UNICEF, 2017). By 2030, global leaders 
have committed to ending preventable deaths of children under 5 years of age, with all 
countries aiming to reduce under-5 mortality to at least as low as 25 per 1,000 live births 
(United Nations Development Program, 2014). 
 
 
3.02. Study objective 
 
To determine the impact of quarterly and bi-annual mass azithromycin administration to 
1-11 month (29-364 days) old infants in rural and periurban Mali on their mortality and 
other health outcomes, on mortality of older children, and to assess the feasibility and 
possible mechanisms of action of the intervention. 
 
 
3.03. Background to and justification for the study 
 
Azithromycin mass drug administration (MDA) has been used for approximately 20 years 
to control trachoma, a blinding eye disease. Over 600 million doses of oral azithromycin 
have been prescribed to children and adults living in an areas of active trachoma program 
(Emerson et al., 2017). In some settings, trachoma control programs with repeated 
treatment of all community members with azithromycin has been associated with 
prevention of other infections, such as malaria, diarrhea and pneumonia (Whitty et al., 
1999; Fry et al., 2002; Coles et al., 2011; Coles et al., 2012; Gaynor et al., 2014; 
Schachterle et al., 2014). Two studies in the same community in Ethiopia, one with a 
case-control design and the other one being a cluster-randomized trial suggested that 
azithromycin MDA might also be associated with reduced under-five mortality (Porco et 
al., 2009; Keenan et al., 2011) 
 
Encouraged by the previous results, two randomized controlled trials were implemented 
recently to test the hypothesis that azithromycin MDA would be associated with reduced 
mortality among 1–59-month-old children in Sub-Saharan Africa. The first study 
(MORDOR) was a placebo-controlled trial in Malawi, Tanzania, and Niger. 
Approximately 1500 clusters were randomly allocated into being either an intervention or 
a control cluster. In the intervention clusters, all 1–59-month-old children were treated 
with a single dose of azithromycin every 6 months for two years; in control clusters 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
children received a respective placebo dose. Neonates, older children and adults were not 
treated.  
 
In the MORDOR sample, azithromycin treatment was associated with a statistically 
significant 13.5% overall reduction in mortality among 1–59-month-old children. The 
difference between intervention and control was approximately 18% in Niger, 6% in 
Malawi and 3% in Tanzania. The differences were largest among 1–5-month-old children 
and after the fourth treatment cycle – but there was not enough power to formally test 
hypotheses about effect modification. Verbal autopsy data suggest that 41% of the deaths 
were due to malaria, 18% to diarrhea or dysentery and 12% pneumonia (Keenan et al., 
2018).  
 
In another trial in Burkina Faso and Mali, researchers tested the impact of combining 
azithromycin MDA to seasonal malaria chemoprevention (SMC, with sulfadoxine-
pyrimethamine and amodiaquine). The intervention was offered monthly to 3–59-month-
old children over three consecutive months. In this sample, there was no difference 
between the trial arms in a combined outcome of hospitalization or death (Chandramohan 
et al., 2019). 
 
Based on earlier results, it thus seems that azithromycin MDA reduces infant or child 
mortality in some, but not in all low-income African settings. An expert committee for 
the World Health Organization has reviewed the data and called for further research, to 
strengthen the evidence-based on azithromycin MDA. Because of the observed 
heterogeneity and possible effect modification by SMC or other co-interventions, further 
trials in new settings are needed in order to make evidence-based public health 
recommendations about the use of this treatment. Because of a fear for antimicrobial 
resistance (AMR), there is also a need to investigate if a more limited target group (1-11 
months) would alleviate the AMR risk but retain the intervention’s impact on mortality. 
Because the MORDOR data suggested that the mortality benefit is concentrated to the 
first three months after the MDA dose (Porco et al., 2019), there is also a need for testing 
the impact of more frequent MDA dosing, on mortality, AMR, and feasibility of the 
intervention, as compared to the previously tested bi-annual treatment of 1–59-month-old 
children. Because of the earlier results suggesting limited additional benefit from 
azithromycin when provided concomitantly with SMC, there is also a need to test the 
impact of bi-annual dosing that is provided quarterly, but only during seasons when SMC 
is not offered. 
 
LAKANA trial has been designed to address the health impacts of quarterly and biannual 
azithromycin MDA when delivered to 1-11-month-old infants in a high-mortality setting 
where malaria is holoendemic but there is also a functioning SMC program in place. The 
trial name (LAKANA) is an abbreviation from “Large-scale Assessment of the Key 
health-promoting Activities of two New mass drug administration regimens with 
Azithromycin. In Bambara, which is the local language in the study area, LAKANA 
means “to protect” or “to be safe”. 
 
 
3.04. Potential public health effects 
 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
The present study may offer a partial solution to a major public health problem in Mali 
and elsewhere in Sub-Saharan Africa. Should the intervention work, it will significantly 
contribute to the options for successfully reducing child mortality which is one of the 
United Nations sustainable development goals. Thus, if the trial provides supportive 
evidence for a positive mortality benefit by azithromycin MDA, it will form the first 
phase of a national public health program in Mali. In phase 2, that would still form a part 
of the currently funded project, an azithromycin MDA program would be rolled out in the 
regions where the trial is implemented, if the government of Mali considers that 
desirable. In phase 3, there could be a national roll-out, the details of and funding for 
which would need to be separately negotiated. 
 
Large-scale use of antibiotics raises always a concern of antimicrobial resistance, which 
could negatively impact clinical outcomes in the communities in which resistance 
emerges. Multiple studies have documented relatively rapid emergence of macrolide 
resistance following mass drug administration with azithromycin (Rogawski et al., 2015) 
but resistance appears to decline quickly after discontinuation of treatment (Sánchez et al. 
1994; Mack et al., 2019) and there is little evidence of AMR spread to individuals who 
have not received the antibiotic (Julia Bielecki, personal communication). Because of the 
concern of possible AMR, we will monitor its prevalence closely in this trial, before, 
during and after the intervention and both among those receiving and those not received 
MDA with the study drugs.  
 
 
3.05. Distribution of the results 
 
The results will be distributed and discussed with the local community, Malian health 
researchers, regional and national level representatives of the Ministry of Health in Mali 
and the World Health Organization. Main findings will be published in international peer-
reviewed journals. 
 
The study material will also be used for post-graduate training of Malian and Finnish 
students. 
 
 
 
4. The study 
 
 
4.01. Objectives of the proposed research 
 
Primary objective: 
1. To evaluate the impact of two azithromycin MDA regimens on infant mortality 
and other health outcomes, when provided in a context a rural West-African high-
mortality context with an ongoing seasonal malaria chemoprevention (SMC) 
program. 
 

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Secondary objectives: 
2. To evaluate the effect of alternative MDA frequencies on AMR and host 
microbiota composition 
3. To investigate the feasibility of alternative azithromycin MDA strategies, 
including economic analysis 
4. To test hypotheses that azithromycin MDA eliminates malaria parasitemia and 
reduces general and intestinal inflammation in asymptomatic children and to 
collect and store biological samples for assessing other possible mechanisms of 
azithromycin effect 
 
 
4.02. Detailed study questions 
 
Detailed questions on mortality (primary objective): 
1.1.Does biannual azithromycin MDA to 1-11 month (29-364 days) old infants, when 
provided quarterly during non-malaria season, reduce their mortality in a Mali-
like setting, in which there is a functional SMC program 
1.2.Does quarterly azithromycin MDA, provided throughout the year (i.e., 4 doses / 
year) to 1–11-month-old infants reduce their mortality in a Mali-like setting? 
1.3.Does quarterly MDA have a bigger mortality effect than biannual MDA? 
1.4.Do the following factors modify the effect of azithromycin MDA on mortality? 
(Exploratory analysis) 
a. Infant age at the time of MDA (1-5 months vs 6-11 months) 
b. Infant weight-for-age, length-for-age, weight-for-length at the time of MDA 
c. Infant sex 
d. Season of MDA dosing (malaria versus non malaria season) and SMC 
implementation in village (SMC given versus SMC not given) 
e. Cluster level coverage of SMC  
f. Cluster level baseline mortality (established at first census) 
g. Cluster and individual level coverage and number of administered azithromycin 
MDA doses 
h. District of residence 
i. Distance from the nearest health facility 
j. Household asset or income index 
k. Household WASH index 
l. National outreach strategy category (standard/advanced) 
i. There is an existing national outreach strategy (Strategie Avancée) 
in place to target communities that have limited access to 
healthcare. Inclusion into that strategy will be treated as a potential 
effect modifier. 
m. Hard-to-reach village status assessed by the field workers 
i. In addition to objective measures (geographical location distance 
to health facilities) and existing national assessment (Strategie 
Avancée), we will conduct a survey among field workers to obtain 
a subjective assessment on how difficult the village is to reach. The 
results will be summarized to a composite variable that will be 
used as a dichotomous variable on whether or not the village is 
“hard-to-reach”. 

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1.5.Does biannual or quarterly azithromycin MDA to 1–11-month-old infants reduce 
mortality among 12–59-month-old children living in the same communities 
(exploratory analysis) 
1.6.Are hard-to-reach communities more likely to experience higher mortality than 
those within closer proximity or easier access to urban locations and health 
facilities (exploratory analysis) 
1.7.Are communities under Strategie Avancée more likely to experience higher 
mortality than those in standard national outreach category (exploratory analysis) 
 
Detailed questions on other health outcomes (primary objective): 
1.8.Does biannual or quarterly azithromycin MDA to 1–11-month-old infants reduce 
the prevalence of ARI, diarrhea, or malaria symptoms among them? 
1.9.Does biannual or quarterly azithromycin MDA to 1–11-month-old infants 
improve their weight gain, linear growth (length), or nutritional status (assess by 
Mid upper-arm circumference MUAC, weight-for-height-Z-score WHZ, Length-
for-age Z-score LAZ, Weight-for-age Z-score WAZ)? 
 
Detailed questions on AMR (secondary objective): 
2.1.Does biannual or quarterly azithromycin MDA to 1–11-month-old infants cause 
an increase in the prevalence of phenotypic azithromycin resistance among S. 
pneumoniae, or E. coli strains isolated from 4-14-month of infants one year after 
the intervention has stopped? 
2.2.What is the impact of biannual or quarterly azithromycin MDA to 1–11-month-
old infants on the prevalence of azithromycin resistance among S. pneumoniae or 
E. coli strains isolated from 49–59-month-old children, who live in the same 
Malian communities but have not received azithromycin MDA, during and after 
the intervention period? 
2.3.Does azithromycin resistant bacteria persist in individual children who received 1-
4 doses of azithromycin 1 year after azithromycin MDA has stopped? 
2.4.Does azithromycin resistant bacteria persist in individual children who received 1-
4 doses of azithromycin 1year after their azithromycin MDA has stopped but 
village MDA intervention has continued? 
2.5.What is the impact of biannual or quarterly azithromycin MDA to 1–11-month-
old infants on the diversity and composition of their intestinal microbiota and 
prevalence of azithromycin resistance genes in intestinal bacteria? 
 
Detailed questions on feasibility including economic analysis (secondary objective): 
3.1 Is scaling-up of azithromycin MDA as a national program to reduce mortality 
among 1–11-month-old infants feasible in Mali according to stakeholders? 
3.2 What are the factors that need to be considered for various scaling-up strategies? 
3.3 What is the level of feasibility, inter-rate reliability, and validity of an instrument 
targeted to capture a subjective assessment of hardness-to-reach amongst the field 
workers? 
3.4 Is there an association between hard-to-reach village status and advanced national 
outreach strategy status (exploratory analysis) 
 
 
Detailed questions on mechanisms of azithromycin action (secondary objective): 

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Protocol version 7.0: 13. November. 2024 
 
4.1 Does azithromycin MDA reduce malaria prevalence and parasite density, and 
increase blood hemoglobin concentration among 1–11-month-old infants? 
4.2 Does azithromycin MDA reduce general inflammation (plasma C-reactive protein 
concentration) among 1–11-month-old infants? 
4.3 Does azithromycin MDA reduce intestinal inflammation (concentration of Alpha-
1-antitrypsine, neopterin, and myeloperoxidase from stool sample) among 1–11-
month-old infants? 
 
 
4.03. Study site and target population 
 
The project will be carried out in rural and peri-urban villages in the Kayes, Kita, and 
Koulikoro regions of Mali, in Sub-Saharan Africa. The regions are situated in western 
Mali, bordered by other Malian regions, Mauritania, Senegal and Guinea. The region has 
235 municipalities (approx. 90% rural), containing close to 3,000 villages. Main means of 
subsistence are farming (cattle and arable), fishing, crop-picking, cotton, peanut and oil 
production. The regions also have goldmining activities (in both formal and informal 
settings). Spoken local languages include Bambara, Malinké, Sarakolé / Soninké / Marka, 
Kassoké, and Peulh. 
 
While the exact mortality rates in the study area are unknown, under-five mortality rate 
(U5MR) is approximately 100 / 1000, infant mortality rate (IMR) approximately 60 / 
1000 and neonatal mortality rate (NMR) approximately 35 / 1000. These figures are 
based on regional 2012-2013 DHS data, when U5MR in Kayes was 96, IMR 60, and 
NMR 34. There was a newer DHS completed in Mali in 2018 and the currently available 
statistics indicate that child mortality has remained at the 2012-2013 levels – hence we 
assume that this is the case also in Kayes, Kita, and Koulikoro.  
 
According to the DHS 2018 report, 19% of U5 children in Kayes are underweight, 26% 
stunted and 9% have severe acute malnutrition. The prevalence of malaria in children 
aged 6-59 months was 12.6%, this prevalence being much higher in rural areas (23%) 
than in urban areas (2%). Seasonal Malaria control is delivered monthly to children aged 
3-59 months between July and October.  
 
The LAKANA trial will be implemented in 7-10 health districts in Kayes, Kita and 
Koulikoro. In the defined study area, there will approximately 1.5 million inhabitants, of 
whom 300,000 are under-five-years old and 60,000 under-one-year old.  
 
 
4.04. Study design 
 
A cluster-randomized, placebo-controlled, double-blinded, parallel-group, three-arm 
clinical trial, with adaptive design. 
 
 

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4.05. Enrollment criteria for participating clusters and participants 
 
The units of intervention allocation, enrollment to the trial, treatment with study drugs, 
and primary outcome measurement will be different, as explained in detail below: 
 
1. The unit of intervention allocation (cluster) will be village (or part of a village). 
Thus, in any one village, all infants will receive the same treatment at each MDA 
round. 
 
2. The unit of enrollment will be a household representative (typically the head of 
household or her / his deputy). An enrolled participant’s household will be visited 
regularly over a period of two years. During the visits, selected household 
members will be interviewed about household composition and eligible infants 
are offered MDA with the study drugs. In a sub-sample of villages, some 
household members may be invited to enrol in sub-studies and additional data 
collection.  
 
3. The unit of treatment with study drugs will be an infant. At each home visit, the 
research staff will assess the eligibility of each child in the household to receive 
study drugs. In consequtive visits, some of the children will be the same who have 
been treated already earlier, whereas others will be new – either because they 
were newly born, they have recetly migrated to the area, or they were not 
available for treatment at the earlier MDA rounds. 
 
4. The unit of primary outcome measurement will be a 3-month time interval, 
starting from the previous home visit and ending with the current one. Any one 
child may contribute 1-4 time-intervals to the main outcome analysis. 
 
Eligibility and permission for participation and treatment will be determined on three 
levels: cluster (village), household, and child. On the cluster level, leaders of eligible 
villages will be asked a permission for study activities to take place in their areas. On a 
household level, a household representative will be asked a consent for trial participation, 
including periodic household visits by the study team and MDA to eligible infants. On 
the child level, eligibility for receiving study drugs will be checked at each of the MDA 
visits, i.e. max 8 times during the trial. 
 
On a cluster level, the inclusion criteria will be: 
1. Location within Kayes, Kita, or Koulikoro region 
2. Considered accessible and safe by the local health authorities and research team 
3. Considered non-urban by the local health authorities and research team 
4. Permission from community leadership 
 
On a household level, the inclusion criteria (for trial enrolment) will be 
1. Location within a cluster that is included in the study 
2. Verbal consent from a head of household or an adult authorized by her / him 
 
On a child level, the criteria for receiving study medication will be: 
1. Residence in a household enrolled in the trial 
2. Age between 29 and 364 days 

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3. Verbal consent from at least one caregiver 
 
On a child level, the exclusion criteria (for not receiving study medication) will be: 
1. Weight below 3.0 kg (3rd centile for healthy 1 month old infants in the WHO 
growth charts) 
2. Known allergy to macrolides, as judged by a caregiver report of the infant 
experiencing an adverse reaction after oral ingestion of medication, that was 
deemed likely to be a macrolide by the interviewing data collector. 
4.06. Trial interventions and control 
 
Participating villages will be randomly allocated to three different intervention groups in 
a ratio of 3 : 2 : 4 (control : azi-quarterly : azi-biannual). Within each village, consenting 
households will be visited quarterly (at 3-month intervals), for nine times. At the first 
eight of these visits, 1–11-month-old eligible infants (age 29-364 days), for whom there is 
a consent for study drug provision, will be weighed and given a single dose of study drug 
(azithromycin mixture or respective placebo mixture). 
 
All study drugs will be coded, so neither the study staff, nor the participants will know 
who gets what. In practice, children will receive the following:  
 
1. Control villages: 
Placebo mixture every three months 
 
2. Azi-quarterly 
Azithromycin mixture every three months 
 
3. Azi-biannual 
Azithromycin mixture at quarterly visits between January 
and June, Placebo mixture at quarterly visits between July 
and December 
 
The dose of the study drug will be 20 mg (0.5 ml) / kg child weight, or an equal volume 
of respective placebo mixture. The study drugs will be given as a single oral dose under 
direct observation, by community volunteers who are involved in other national mass 
drug administration campaigns in the same villages.  
 
Figure 1 shows a schematic illustration of ingredients in quarterly study drugs, in the 
three study groups: 
 
  
A = Azithromycin, P = placebo 
4.07. Outcome variables 
 
Primary efficacy outcome: 
1. Mortality (deaths / 1000 years at risk) among children who were 1-11 month (29-
364 days) old at the time of study drug administration. 
Apr-Jun 
2020
Jul-Sep 
2020
Oct-Dec 
2020
Jan-Mar 
2021
Apr-Jun 
2021
Jul-Sep 
2021
Oct-Dec 
2021
Jan-Mar 
2022
Apr-Jun 
2022
Jul-Sep 
2022
Oct-Dec 
2022
Control
P
P
P
P
P
P
P
P
P
P
P
Azi-quarterly
A
A
A
A
A
A
A
A
A
A
A
Azi-biannual
A
P
P
A
A
P
P
A
A
P
P

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2. As an exploratory analysis, we will assess the following factors as potential effect 
modifiers to the azithromycin impact on mortality among the 1–11-month (29-364 
days) olds 
a. Infant age at the time of MDA (1-5 months vs 6-11 months) 
b. Infant weight-for-age, length-for-age, weight-for-length at the time of 
MDA 
c. Infant sex 
d. Season of MDA dosing (malaria versus non malaria season) and SMC 
implementation in village (SMC given versus SMC not given) 
e. Cluster level coverage of SMC 
f. Cluster level baseline mortality (established at first census) 
g. Cluster and individual level coverage and number of administered 
azithromycin MDA doses 
h. District of residence 
i. Distance from the nearest health facility 
j. Household asset or income index 
k. Household WASH index 
l. National outreach strategy category (standard/advanced) 
m. Hard-to-reach village status assessed by the field workers 
 
Secondary efficacy outcomes: 
1. Prevalence of diarrhea, ARI, malaria, or other illnesses in the 14-day period 
before MDA visits 
2. Mean weight-for age, length for age, weight-for-length, and mid upper arm 
circumference, and prevalence of moderate to severe stunting (LAZ < -2 / LAZ < 
-3) and moderate to severe wasting (WLZ < -2 / WLZ < -3) at 6-8 and 12-14 
months of age. 
3. Mortality (deaths / 1000 years at risk) among children who were 12–59-month-old 
when the latest azithromycin MDA took place in their village of residence.  
a. This will be analyzed since azithromycin MDA could elicit herd 
protection that could theoretically extend beyond the actual recipient 
population of the treatment. 
 
Outcomes related to antimicrobial resistance: 
1. Phenotypic macrolide resistance among E. coli strains isolated from stool samples 
or S. pneumoniae strains isolated from nasopharyngeal swabs among 4–14-
month-old children, at one year after the MDA intervention has stopped (36 
months after baseline). 
a. As descriptive information, we will provide the same information from a 
smaller group of children at baseline and when the intervention has 
continued for 12 and 24 months (i.e., after 4 or 8 rounds of MDA). 
b. For all isolates of azithromycin resistant E. coli or S. pneumoniae, we will 
also determine of phenotypic AMR against other antibiotics categorized 
by the World Health Organization (WHO) into "Access" group and/or that 
may have overlapping mechanisms: meropenem, ceftriaxone, 
ciprofloxacin, ampicillin, co-trimoxazole, and gentamicin for E.Coli, and 
oxacillin, erythromycin, vancomycin, ciprofloxacin, ampicillin, co-
trimoxazole for S. pneumoniae. 

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2. Phenotypic macrolide resistance among E. coli strains isolated from stool samples 
or S. pneumoniae strains isolated from nasopharyngeal swabs among 49–59-
month-old children, at 24 months after cluster enrollment to the trial, i.e. when the 
intervention has continued for 24 months (after 8 rounds of MDA). 
a. 
As descriptive information, we will provide the same information from a 
smaller group of children at baseline, at 12 months after enrolment (4 
rounds of MDA), and at one year after the MDA intervention has 
stopped 
b. In here, we are primarily interested in the older age-group, since earlier 
research suggests that AMR prevalence will increase among the treated 
individuals, but we expect AMR not to spread to non-treated individuals. 
c. In a two-year MDA and follow-up period, 49–59-month-old children will 
never have received any azithromycin as part of the trial. 
d. For all isolates of azithromycin resistant E. coli or S. pneumoniae, we will 
also determine of phenotypic AMR against other antibiotics categorized 
by the World Health Organization (WHO) into "Access" group and/or that 
may have overlapping mechanisms: meropenem, ceftriaxone, 
ciprofloxacin, ampicillin, co-trimoxazole, and gentamicin for E.Coli, and 
oxacillin, erythromycin, vancomycin, ciprofloxacin, ampicillin, co-
trimoxazole for S. pneumoniae. 
3. Genetic markers of azithromycin and other antibiotic resistance (resistome) of 
intestinal and nasopharyngeal microbiota among 4–14-month-old children, at one 
year after the MDA intervention has stopped. 
a. As descriptive information, we will provide the same information from a 
smaller group of children at baseline and when the intervention has 
continued for 12 and 24 months. 
b. We are primarily interested in the post-intervention situation, since earlier 
research suggests that AMR prevalence will increase among the treated 
individuals soon after azithromycin treatment, but we expect the 
prevalence to go down after the intervention. 
4. Carriage of bacteria with azithromycin resistance in children who received 1-4 
rounds of MDA and who live in villages that have stopped MDA intervention 1 
year prior to sampling (children who are 15-26 month-old at visit 11)  
a. As descriptive information, we will provide the same information from a 
smaller group of children one year after the MDA intervention has 
stopped. 
b. Earlier research has shown that AMR prevalence will increase among 
treated individuals. How and if this prevalence persists in these treated 
children 1 year after their treatment has finished and the MDA 
intervention ended in the villages they live is currently not known. We will 
assess phenotypic macrolide resistance and resistance against other 
antibiotics categorized by the World Health Organization (WHO) into 
"Access" group and/or that may have overlapping mechanisms: 
meropenem, ceftriaxone, ciprofloxacin, ampicillin, co-trimoxazole, and 
gentamicin for E.coli, and oxacillin, erythromycin, vancomycin, 
ciprofloxacin, ampicillin, co-trimoxazole for S. pneumoniae. 
c. We will assess genotypic markers of resistance and changes in the 
intestinal microbiome in children previously treated with azithromycin and 
we expect that the microbiome in these children has recovered 1 year post 

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Protocol version 7.0: 13. November. 2024 
 
treatment and is similar to the microbiome of children in the placebo 
group. 
 
5. Carriage of bacteria with azithromycin resistance in children that received 1-4 
rounds of MDA and who live in villages (tertiary villages in Kati region) that are 
still on MDA intervention 1 year prior to sampling (children that are 24-35 
month-old at visit 9) 
a. As descriptive information, we will provide the same information from a 
smaller group of children 1 year after their individual treatment ended but 
the villages they live in are still on MDA intervention. 
b. Earlier research has shown that AMR prevalence will increase among 
treated individuals. How and if this prevalence persists in these treated 
children 1 year after their treatment has finished but the villages they live 
in are still on MDA intervention is currently not known. We will assess 
phenotypic macrolide resistance and resistance against other antibiotics 
categorized by the World Health Organization (WHO) into "Access" 
group and/or that may have overlapping mechanisms: meropenem, 
ceftriaxone, ciprofloxacin, ampicillin, co-trimoxazole, and gentamicin for 
E.coli, and oxacillin, erythromycin, vancomycin, ciprofloxacin, ampicillin, 
co-trimoxazole for S. pneumoniae. 
c. We will assess genotypic markers of resistance and changes in the 
intestinal microbiome in children previously treated with azithromycin and 
we expect that the microbiome in these children has recovered 1 year post 
treatment and is similar to the microbiome of children in the placebo 
group. 
 
Outcomes related to the mechanism of azithromycin activity 
1. Blood C-reactive protein concentration 
2. Blood malaria parasitemia, and hemoglobin concentration 
3. Fecal neopterin, myeloperoxidase, and alpha-1-antitrypsin concentrations 
 
Safety outcomes: 
1. Incidence of serious adverse events (SAE) within 14 days of study drug 
administration.  
2. Incidence of adverse events (AE) within 14 days of study drug administration.  
4.08. Data collection sites for various outcomes 
 
Mortality and serious adverse event (SAE) data will be collected, and mortality-related 
questions answered using data from all the included 1150 villages (clusters) . 
 
Addressing the other study questions will require more study personnel and additional 
equipment and infrastructure but a smaller sample size. Hence, we will answer them in a 
sub-sample of 60 villages, housing approximately 3,000 infants and located around four 
selected health centers close to the city of Kita (secondary outcome sample). Working in 
and from there health centers, there will be special teams of research personnel, that will 
collect information on the following outcome variables: 
1. Mortality, as in all the other villages (see chapters 4.12 and 4.13 for details) 
2. Morbidity for acute infections (see chapter 4.14 for details) 

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Protocol version 7.0: 13. November. 2024 
 
3. Growth and nutritional status (chapter 4.15) 
4. Antimicrobial resistance (chapter 4.16) 
5. Mechanism of azithromycin action (chapter 4.17) 
6. Feasibility, acceptability and equity of intervention (chapter 4.18) 
7. SAE and adverse events (AE) (chapter 4.19) 
 
Whilst these 60 are in a more confined area than those constituting the primary outcome 
sample, they represent a similar, mostly rural population. Hence, there is no a priori 
reason to expect non-representativeness of the secondary outcome sample. To assess the 
representativeness, we will, however, compare selected baseline characteristics (such as 
child age, sex, weight-for-age, and household asset index) in the primary and secondary 
outcome sample before the statistical analysis. A dataset that excludes data from the 
secondary outcome sample will be used for a sensitivity analysis on the interventions 
effect on mortality. 
 
Figure 2 shows a schematic illustration of the sample structure. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In addition to the above mentioned 60 villages in Kita, we intend to collect AMR samples 
and mechanistic study samples from infants and children living in a similar number of 
villages closer to Bamako, i.e., Koulikoro or Kati (tertiary AMR sample). For the 
mechanistic study in the tertiary sample, we will collect the biological samples during the 
rainy season (peak malaria transmission season). In addition to the AMR and mechanistic 
study samples, we will consider growth outcomes measurements (anthropometry) in the 
tertiary sample if the number of those measurements proved smaller than expected in the 
secondary sample villages. 
 
4.09. Schedule and procedures for participant enrollment and follow-up 
 
During a preparation phase, all villages / clusters in the included health districts of the 
three study regions will be listed and mapped. Scientists in the study team, working with 
the local health professionals, will review the map and exclude areas and villages that are 
considered urban, unsafe, inaccessible or unstable in population (in gold-mining areas, 
for example), to come up with a final list of invited clusters. 
 
Senior-level study team supervisors, accompanied by local community volunteers and 
(1150 clusters, approx. 154 heath facilities) 

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local health service representatives, will visit leadership within each village / cluster, 
brief them about the trial (Appendix 1a) and invite them to participate. Villages that allow 
LAKANA activities to take place will be randomly allocated (in a ratio of 3:2:4) to three 
different intervention schemes: control, azi-quarterly, and azi-biannual . Village level 
permission will be given verbally and documented in writing in the project notebooks 
(Appendix 1b). 
 
The included villages will be visited at quarterly intervals by GCP-trained study staff, 
accompanied by local community volunteers. At each round, there will be a two-week 
period, during which all households in the village will be enlisted, located, and visited if 
possible. At the first visit to each household, household representatives are briefed about 
the study (Appendix 2a) and invited to participate in the trial, including eight MDA 
treatments and a two-year follow-up. Those agreeing will be revisited eight more times at 
quarterly intervals. Households participating in an AMR sub-study (see chapter 4.15) will 
be visited one more time 12 months later. 
 
At the enrollment visit, there will be a census of all household members and an interview 
with a household representative about some baseline socio-economic and other variables 
(including health facility that they normally use) as well as births and child deaths in the 
preceding year (to establish neonatal, infant, and under-five mortality rates at baseline). 
Children between 1 and 11 months of age will be assessed for MDA eligibility, weighed, 
and provided with study drug if eligible. At the subsequent seven visits, there will be an 
interview about recent births, child deaths, and health facility visits and again a study 
drug MDA to 1-11-month (29-364-day) old infants. At the 9th visit, there will be just the 
interview, but no MDA. At each of the visits 1-9, we will also ask about MDA treated 
infants’ exposure to SMC (as a potential effect modifier) and document their EPI 
vaccination dates (from their vaccination card, as a marker of health service utilization). 
 
There will be no upper or lower limit in the number of participants / study village, i.e. all 
households and infants who can be enrolled or treated with study drugs during any MDA 
round will be included in the study. 
 
Infant age will be calculated with a computer, based on dates of visit and child’s date of 
birth. Date of birth will be obtained primarily from a child health card, secondarily from 
caregivers’ information (if exact date is reportedly known) and tertiarily estimated with 
the use of a time-bound event calendar. 
 
Participating districts and villages will be enrolled into the study in a rolling manner over 
a period of 12 months and visited at regular 3-month intervals thereafter for 24 months. 
This will make logistics easier and reduce the risk of obtaining a biased estimate of 
azithromycin effect, in case this effect was modified by season and also by the order of 
MDA round. To shorten the trial period by six months, the last enrolled villages will be 
followed up only for 7 MDA rounds (30% of villages) or 6 MDA rounds (20% of 
villages). 
 
As explained in general in chapter 4.08 and in detail in chapters 4.13 – 4.18, there will be 
additional data collection on several secondary outcomes in selected clusters and health 
centers. These data collections are timed in a way that maximises the possibility of 
observing a treatment effect and spreads the activities relatively evenly over the entire 

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trial period, thus maximising also staff efficiency.  
 
Figure 3 shows an outline of the follow-up of individual LAKANA trial participants.  
 
  
Additionally, we will collect samples at visit 11 from 15-26-months old children and at 
visit 9 in the Koulikoro/ Kati (tertiary AMR sample) region from 24-35-months old 
children. 
 
4.10. Expected recruitment and loss-to-follow-up rates 
 
The planned number of clusters (villages) to be included in the trial is approximately 
1150, situated around approximately 154 health facilities. These villages are planned to 
be enrolled in the trial over a period of 18 months, i.e., 78 weeks. Hence, every week 
there will be on average 15 new clusters enrolled to the trial, situated around 1-3 new 
health facilities. The final number of clusters will depend on the cluster size; the village 
enrolment will be stopped before 1150 villages if 35,650 infants have been treated on 
MDA1 prior to that. 
 
In each cluster, there are an estimated average of 35 infants of 1-11 months (29-364 days) 
of age. We assume to be able to obtain consent and provide study drugs to 31 (>85%) of 
them at each MDA round. Hence during the enrollment phase, we expect to provide 
MDA to 800 infants per week in the first three months, then 1600, 2400, and 3200 infants 
per week for three subsequent 3-month periods. We will then continue to treat 
approximately 32 00 infants / week for the next 12 months and then gradually stop 
treatments in the subsequent 9 months. 
 
At each of the eight quarterly rounds of MDA, we thus expect to provide MDA to 
approximately 41,500 infants. At the first visit, all the treated infants will be new to the 
trial, on the subsequent visits, approximately 25% will be new infants, others will have 
received the study drugs already earlier. The maximum number of study drug treatments 
to any single individual will be four.  
 
Since each time point will be assessed cross-sectionally, it will not be possible to 
calculate a traditional loss-to-follow-up rate. At each time, we expect to be able to get 
vital status information from approximately 80%, who were given study drug MDA at the 
previous home-visit. This proportion is based on prior experience from the Mordor trial, 
in which there was a 13% loss to follow-up at biannual home-visits. 
 
 
1 2 3 4 5 6 7 8 9 # # # 1 2 3 4 5 6 7 8 9 # # # 1 2 3 4 5 6 7 8 9 # # # 1 2 3 4 5 6 7 8 9 # # #
Mortality
Full sample
A - F
01-11 mo
32,000
1
2
3
4
5
6
7
8
9
Morbidity
4 CSCOMs
A
01-11 mo
1,350
Antimicrobial resistance
Participants & contacts
4 + 2 CSCOMs
A
04-14 mo
49-59 mo 1350+1350 1
5
9
11
Mechanism of action
4 CSCOMs
A
06-08 mo
12-14 mo 1350+1350
3
4
5
6
Growth
4 CSCOMs
A
06-08 mo
12-14 mo 1350+1350
3
4
5
6
Feasibility
4 CSCOMs
A
01-11 mo
1,350
2
10
Safety and adverse events4 CSCOMs
A
01-11 mo
1,350
1
3
4
5
6
11
Year 1
Year 2
Year 3
Year 4
Outcome
District
Sample
Age 
group
Number 
of 
children

Trial protocol: LAKANA 
Page 26 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
4.11. Purchase, formulation, storage, and distribution of study drugs 
 
The study drugs (azithromycin and placebo) will be donated by Pfizer Inc (New York 
City, NY, USA), that will manufacture and package them under good manufacturing 
practice (GMP) conditions and ship them to Mali.  
 
The drugs will be packed in non-opaque, plastic bottles. Each bottle will contain the same 
amount of dry powder, either including 1.2 g of azithromycin or respective amount of the 
base powder.  
 
A data manager at RTI International, an external partner for data support services, will 
randomly allocate 8 letter codes to azithromycin and 10 other letter coders to placebo and 
transmit the trial code to a pharmacist at Pfizer. Study drugs will be labelled with these 
letter codes. For each cluster, we will use bottles with two letter codes, one for visits 
between January and June and the other one for visits between July and December. Thus, 
there will be a total of 9 two-letter study drug regimens, of which 3 will be allocated to 
control (both letter codes for placebo), 2 will be for azi-quarterly (both letter codes for 
azithromycin) and 4 will be for azi-biannual (one letter code for placebo, the other one 
for azithromycin). 
 
During shipment and storage at the study site, the study drug bottles will be stored in 
locked cabinets, in a dry place with a temperature between 15oC and 30oC. 
 
For consumption, a drug bottle will be reconstituted with 15 ml of clean (commercial 
bottled) water to make 30 ml of study drug suspension. The reconstituted mixture will 
thus contain either 40 mg / ml azithromycin (active drug) or no azithromycin (placebo). 
 
The dose given to eligible children will be 20 mg / kg, i.e. 0.5 ml / kg, rounded up to the 
nearest 0.2 ml. A data collector will measure and give the dose to study participants with 
a single-use sterile syringe, under direct observation. There will not be any planned 
observation period of the infant immediately after the study drug administration, but the 
caregivers are advised to contact the study team if anything unusual happens. If a study 
team member becomes aware that an infant has vomited soon after the study drug 
ingestion (within approximately 15 minutes), they will give a new, similar-size dose to 
the infant in question.  
 
At any village (cluster), all infants will receive study drug of the same letter code. Hence, 
a bottle can be reconstituted and used for several participants on the same day. An 
expected mean dose will be 3 ml, i.e. one bottle may be enough for up to 10 participants. 
For each MDA round, we would thus need an average of 2,500 bottles of placebo and 
2,000 bottles of azithromycin. Given the 8 MDA rounds and an assumption of 80% 
wastage in storage (with several letter codes for each product and a geographically a wide 
operation), we estimate to need 100,000 bottles of placebo and 80,000 bottles of 
azithromycin for the whole trial. 
 
At the end of each day, the remaining reconstituted study drugs will be brought back to 
the study office. At the study office, the left-overs will be discarded, according to national 
instructions. 
 

Trial protocol: LAKANA 
Page 27 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
 
4.12. Data collection on infant and child mortality 
 
At the enrollment visit, a data collector hired by the the study team will interview a 
household head to collect information on household composition. S/he will record the 
houshold members in a password-protected and encrypted electronic trial database, to 
which s/he can refer to and which s/he will update during subsequent visits. The database 
will include the following information on each member of the household: Sex, name, date 
of birth, age, date of start of follow-up (enrollment of household or in-migration), date of 
end of follow-up (death, withdrawal, or out-migration) 
 
At each of the subsequent visits, the data collector will check the vital status (alive, dead, 
out-migrated, or unkown) of the previously registered household members and record any 
new births and in-migrations. If a child death - is noticed, there will be a special set of 
questions on the date and cause of death. Cause of death will be classified into trauma, 
acute illness of less than two weeks duration, prolonged illness of more than two weeks 
of duration, or other, i.e. no extensive verbal autopsies or other cause of death 
ascertainment will be done. Valid vital status information can be obtained from a parent 
or other caretaker of the child who lives in the same household; if such a person is not 
available, the information will be considered missing. 
 
Mortality will be expressed as deaths / 1000 years at risk. Each 3-month interval will 
contribute to mortality calculation. Children will be included in the analyses if they were 
present in the household at the beginning of the interval and their vital status (dead or 
alive) was known and provided to the study team at the subsequent visit, by an eligible 
adult (see above for eligibility criteria). Time in follow-up will be the number of days 
between the beginning of the interval and death, outmigration, or end of interval, 
whichever came first. All mortality data i.e. from both the primary outcome sample and 
the secondary outcome sample will be used for the primary mortality outcome analysis. 
 
Mortality rates will be calculated separately for 29-364 days old infants (the primary 
target population for the intervention) and for 12.00-59.99 month old children (who have 
not themselves received the MDA intervention). The indicated figures refer to the 
subject’s age at the beginning of the interval. 
 
 
4.13. Adaptive design: possible changes after an interim analysis 
 
An interim analysis will be conducted when approximately 60% of the planned 3-month 
time-intervals have been completed. The analysis results will be seen and reviewed only 
by the trial data safety and monitoring board (DSMB) that will advise the study team on 
trial continuation, according to its charter. Based on its adaptive design, the trial conduct 
will be altered in case the interim analysis gives either of the following results: 
 
1. There is evidence of a mortality benefit in both of the azithromycin groups as 
compared to the placebo group but no statistically significant difference between 
the two azithromycin groups. 

Trial protocol: LAKANA 
Page 28 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
a. In this scenario, the team will drop the placebo arm and re-randomize the 
previous placebo-clusters into either of the two azithromycin groups, at 1:1 
allocation ratio. All trial participants will be informed about the change and 
a new consent will be requested for participation in the revised trial. 
b. Before implementation, the participant information leaflet and an amended 
consent form will be submitted to the appropriate ethics committees and 
institutional review boards (IRB) for review and approval. 
c. Subject to endorsement by the trial data safety and monitoring board 
(DSMB) before the onset of data collection, the team proposes to consider 
a mortality difference in an interim analysis statistically significant if a 2-
sided p-value is smaller than 0.001 (Peto et al., 1976). 
d. After the results from the azi-quarterly and azi-biannual groups comparison 
are available, the team will offer to work with the Malian Ministry of 
Health on implementation of a public health MDA intervention in Kayes, 
Kita, and Koulikoro and possibly Bougouni and Segou regions of Mali, 
choosing the MDA regimen (quarterly or biannual) based on the trial data. 
 
2. There is evidence of a mortality benefit in one or both of the azithromycin groups 
and also a statistically significant difference between the two azithromycin 
groups. 
a. In this scenario, the provision of the study MDA will be stopped, and the 
team will offer to work with the Malian Ministry of Health on 
implementation of azithromycin MDA in Kayes, Kita, and Koulikoro and 
possibly Bougouni and Segou regions of Mali, choosing the MDA 
regimen (quarterly or biannual) based on the trial data. 
 
A separate trial (SANTE) in Sikasso Region is evaluating the benefits of azithromycin 
provided to pregnant women and to infants at the 6-week EPI visit. The combined results 
of the SANTE trial and the newly proposed trial will provide important evidence for 
decision-making around the optimal strategy for delivering azithromycin for child 
survival in the whole of Mali. 
 
The adaptive design is illustrated in Figure 4 below.  
 
 

Trial protocol: LAKANA 
Page 29 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
  
 
 
4.14. Data collection on infant morbidity 
 
Earlier trials and other studies have suggested that azithromycin MDA might also have an 
impact on child morbidity. Whilst it would be difficult to assess morbidity reliably on an 
individual level in all LAKANA participants, we intend to collect morbidity related 
information among infants who live in the 60 villages selected for additional outcome 
assessment (secondary outcome sample). These villages are served by four health centers, 
where we will establish a LAKANA office and post research staff for the additional data 
collection. 
 
During the normal MDA home visits in these selected villages, the LAKANA team will 
ask if a child has been ill in the preceding 14 days, with fever with respiratory symptoms 
(acute respiratory infection, ARI), fever without respiratory symptoms (proxy for 
malaria), diarrhea, or any other symtoms. These questions will be asked about each 4-14 
month old infants and children, i.e. those who were eligible to receive the study drug 
intervention at an earlier MDA round.  
 
From the collected data the team will calculate incidence of various types of illness 
episodes in the three study arms. The team will then calculate relative risks for the 
illnesses in azi-quarterly and azi-biannual groups, as compared to the control group.  
 
 
4.15. Data collection on infant growth and nutritional status 
 
A relatively recent meta-analysis suggested that antibiotic treatment of children in low-
income countries might improve their linear growth (Gough et al., 2014). The review did 
not include enough studies on infants, so the question of antibiotic effect on infant growth 
is still open. It is, however, possible, that azithromycin might also promote growth in this 
age group (in which length and weight gain is faster than among older children), and 
hence it justifiable to study growth as a secondary outcome in a mortality trial.  
 

Trial protocol: LAKANA 
Page 30 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
The sub-sample will be formed from children, who reside in the 60 villages around the 
four selected health centers (secondary outcome sample). In these villages, data collectors 
will take anthropometric measurements from 6-8 and 12-14 month old children on study 
visits 6, 7, 8, and 9, i.e. at 15, 18, 21, and 24 months after the enrollment of the village 
into the trial. At these visits, the 6-8 month-old and 12-14 month old children will have 
received two and four doses of study drugs, respectively, unless they migrated into the 
village later, were absent, unlocated or declined study drug administration. Thus this 
sampling frame ensures maximal exposure to the study drug at size assessment. 
 
At each time point, we expect to measure a mean of 7.5 children / village, i.e. the total 
sample size will be 1800 children, i.e. approximately 600 per group. This sample size will 
provide the study with 80% power to detect difference of approximately 0.2 SD units in 
anthropometric indices, which is a typical achievement in nutritional supplementation 
trials (Dewey and Adu-Afarwuah, 2008). 
 
Measuments taken from the sub-sample include length, weight, and mid upper arm 
circumference (MUAC). Length will be assessed using a commercial length board 
(ShorrBoard®, WEIGH AND MEASURE, LLC, Olney, Md, USA) and recorded to the 
nearest 1 mm. Weight will be assessed using an electronic infant weighing scale (SECA 
354) with reading increments of 10g. MUAC will be measured with non-stretchable 
plastic insertion tapes and the results will be recorded to the nearest 1 mm. All 
measurement will be done in triplicate. 
 
The data collectors measuring anthropometrics will be trained and standardized prior to 
beginning of data collection, modified from the procedures used in the WHO 2006 
growth standard study (WHO, 2006). Standardization sessions will be repeated every 6 
mo during data collection. We will calculate weight-for-age (WAZ), length-for-age 
(LAZ), weight-for-length (WLZ), and arm circumference-for-age z-scores using the 
WHO 2006 Child Growth Standards. We will also calculate the percentage of children 
who are moderately or severely stunted (LAZ < -2 / -3), or moderately or severely wasted 
(WLZ< -2 / -3). Those with wasting will be referred for nationally recommended 
diagnostics and treatment. 
 
 
4.16. Data collection on antimicrobial resistance 
 
One of the major concerns in the use of azithromycin MDA is the possibility of 
increasing AMR, resulting in the loss of mortality benefits, and possibly even increasing 
health problems. As explained in chapter 3.03, macrolide resistance has earlier been 
observed in the treated individuals soon after azithromycin MDA, but there is relatively 
little data on longer follow-up or spread of AMR to unexposed populations. In 
LAKANA, we will therefore monitor prevalence of azithromycin resistance before, 
during and after the intervention and both among children receiving and those not 
receiving MDA with the study drugs. 
 
To allow comparability to national and international AMR statistics, we will primarily 
study AMR with a traditional phenotypic culture method, isolating individual colonies of 
selected indicator bacteria (S. pneumoniae and E. coli) from children and assessing 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
bacterial growth on a Petri-dish in the presence or absence of azithromycin. To maximise 
comparison of these surveillance cultures with antimicrobial resistance results from other 
azithromycin MDA trials (such as the AVENIR trial in Niger), details of sample 
collection and processing methods will be harmonised between the different studies.  
 
Studying individual colonies of selected indicator bacteria is a widely used approach for 
AMR assessment, but it gives no information on possible antibiotic effects on other 
colonies of the same species or any of the other bacterial species (some of which may be 
clinically much more important than the indicator bacteria). We will therefore also 
employ genetic methods to study microbiota composition and the presence of molecular 
markers of AMR in host bacteria. As sample collection for the AMR component will take 
three years and molecular methods are rapidly developing, we will store samples for 
these genetic (and possible other) analyses until the completion of data collection and 
choose the exact molecular methods only at that time point. Similar to the indicator 
bacteria analyses, these sample collection and processing for these studies will be 
harmonised with the AVENIR trial in Niger. 
 
In the first phase, we will thus address the following questions: 
1. What is the impact of biannual or quarterly azithromycin MDA to 1-11 month 
(29-364 days) old infants on the prevalence of phenotypic azithromycin resistance 
among S. pneumoniae or E. coli strains isolated from 4–14-month-old children, 
who have received 1-4 rounds of azithromycin MDA? 
2. What is the impact of biannual or quarterly azithromycin MDA to 1-11 month 
(29-364 days) old infants on the prevalence of phenotypic azithromycin resistance 
among S. pneumoniae or E. coli strains isolated from 49–59-month-old children, 
who live in the same Malian communities but have not received azithromycin 
MDA?  
3. What is the impact of biannual or quarterly azithromycin MDA to 1-11 month 
(29-364 days) old infants on the prevalence of phenotypic azithromycin resistance 
among S. pneumoniae or E. coli strains isolated from 15-26-month-old children, 
who have received 1-4 rounds of azithromycin MDA and live in a village that 
stopped azithromycin MDA 1 year before? 
4.  What is the impact of biannual or quarterly azithromycin MDA to 1-11 month 
(29-364 days) old infants on the prevalence of phenotypic azithromycin resistance 
among S. pneumoniae or E. coli strains isolated from 24-35-month-old children, 
who have received 1-4 rounds of azithromycin MDA 1 year before and who live 
in a village that continued azithromycin MDA? 
 
To ascertain if clinical disease is being influenced by azithromycin MDA, we will also 
study AMR in bacteria isolated from stools with children presenting at any of the four 
selected health facilities because of dysentery (bloody stools) or blood, urine, or 
cerebrospinal fluid of 1–11-month-old infants or older children hospitalized in the nearby 
Kita District Hospital because of a severe, septic illness. For this purpose, we will 
establish a basic microbiology laboratory in this hospital and support its operations 
during the LAKANA trial. We have chosen Kita for this purpose, because it is the largest 
hospital in the Kita region and because we will establish our LAKANA biospecimen 
processing and storage laboratory in this hospital. Kita is also relatively near Bamako, 
which facilitates support and oversight by the CVD-Mali laboratories and the LAKANA 
research team. 

Trial protocol: LAKANA 
Page 32 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
 
In the main CVD-Mali laboratory in Bamako (level 3) or Kita laboratory (level 2), the 
study team will perform microbiological analyses that can be fed back to clinicians to 
support clinical management of infections. The clinical physicians can then target their 
treatment based on the microbiological findings on bacterial type and basic bacterial 
characterization, such as Gram stain. In addition to this simple analytical laboratory, the 
team may establish a more basic field laboratory (level 1) in a health facility in 
Koulikoro. At this facility, the study team can process, and store collected AMR samples 
and perform initial bacterial growth analysis using an automated suspension cultivation 
method. All AMR analyses will be performed in the main CVD-Mali laboratory in 
Bamako (level 3). In additional bacterial culture, the team will also perform resistome 
analysis and other molecular AMR testing from the isolated bacterial colonies, with 
methods that are harmonized with those of the AVENIR trial in Niger. 
 
Our assumption is that among the treated children, AMR prevalence will increase during 
the intervention and return to pre-study levels after the intervention has stopped. Among 
the older children, who live in the same treatment communities but who have not 
themselves received any MDA, we expect no change in AMR prevalence. The assumed 
AMR prevalence over time in the two samples is shown in Figure 5 below. 
 
 
The hypothesis to be tested from the 4–14-month-old children is that azithromycin does 
not cause a sustained increase in AMR, i.e., that AMR prevalence will not be higher in 
the azithromycin clusters than the control clusters at one year after the intervention. For 
the older children (who received no MDA), the respective hypothesis is that AMR 
prevalence will not be higher in the azithromycin clusters than the control clusters at any 
point, not even at the peak of the intervention – i.e at 12-24 months from the study start. 
Thus, for AMR, we have a non-inferiority hypothesis – i.e., that azithromycin MDA can 
be used to reduce infant mortality without a clinically significant increase in AMR 
prevalence. In an exploratory analysis, we will also compare AMR prevalence in the two 
azithromycin groups, to investigate if quarterly MDA selects for more AMR that the 
biannual MDA. 
 
The suggested set-up will allow us to describe concomitant time trends in the prevalence 
of AMR in indicator bacteria isolated from healthy children, the incidence of septic 

Trial protocol: LAKANA 
Page 33 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
diseases, and the prevalence of AMR in bacteria that are causing severe disease. This will 
facilitate understanding about the clinical significance of AMR, possibly caused by the 
MDA intervention. 
 
For hypothesis testing on the prevalence of AMR in indicator bacteria, we will need a 
total of 1,350 children. These children will be identified in the 60 villages around the four 
health centers selected for secondary outcome data collection in Kita. All 4–14-month-
old and 49-59 month old children in this sub-sample will be invited to provide biological 
samples for AMR analyses at enrollment (visit 1) and at 12 months (visit 5), 24 months 
(visit 9) and 36 months (visit 11) after the inclusion of the village into the study.  
 
To facilitate the AMR sample collection before study drug administration, the team 
making home visits in this sub-sample of clusters will include a study nurse. Before any 
MDA, this nurse will collect one nasal and three rectal swabs from the study infants, in a 
tent set up in the village that is being visited.  
 
To assess whether AMR carriage persists on an individual level we will sample children 
at the final time point (visit 11) who have received 1-4 MDA doses and who live in a 
village that stopped MDA intervention 1 year earlier (children at 15-26 month-old). 
Additionally, we will sample children (24-35 months old) at visit 9 (Koulikoro/Kati 
region) who have received 1-4 MDA doses 1 year earlier, but live in a village that 
continued MDA intervention. 
 
After collection, the nasopharyngeal swabs will be placed in STGG media (skim milk, 
tryptone, glucose, and glycerin), one rectal swab will be placed in Cary-Blair media, the 
second rectal swab will be placed into will be placed in DESS media 
(DMSO/EDTA/saturated sodium chloride) and the third rectal swab will be frozen 
without preservatives in a cryovial. The third rectal swabs will be flash frozen in dry 
shippers containing liquid nitrogen that has been absorbed in the container linings. Other 
samples will be placed in a +4oC cooler box. All samples will be transported within 2-4 
hours to a study laboratory in Kita or to the main laboratory in Bamako, where a 
laboratory technician will process them and store them at -80oC: From the Kita 
laboratory, samples will be periodically transported to the main CVD-Mali laboratory for 
processing.  
 
Nasopharyngeal swabs will be primarily cultured on chocolate agar with an optochin 
disc, to identify pure colonies of Streptococcus pneumoniae. Rectal swabs will be 
primarily cultured on MacConkey plates, to identify pure clones of Escherichia coli. 
Isolated colonies will then undergo phenotypic AMR analysis with bacterial culture on 
Petri-dishes supplied with Azithromycin containing antibiotic strips (E-test).  
 
In the first phase, we will determine azithromycin sensitivity from all the samples 
collected from 4–14-month-olds at 24 months and from 49-59 month olds at 36 months 
(n = 1,350 per time point and age group) and from 1/3 of the samples at the other time-
point (for descriptive purposes, not for hypothesis testing). We will also determine 
azithromycin sensitivity from samples collected from 15-26-month-olds at 36 months 
(Kita region) and 24-35-month-olds at 24 months (tertiary AMR samples). We will then 
calculate AMR prevalence for in S. pneumoniae and E. coli, for each of the time points 
and each of the three trial arms separately. At each time point, we will calculate the 

Trial protocol: LAKANA 
Page 34 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
difference (95% confidence interval, CI) in AMR prevalence between the control and azi-
biannual clusters on one hand and between the control and azi-quarterly clusters on the 
other. The pre-set non-inferiority margin is 10 %-points, i.e. if the entire 95%CI of the 
difference in less than 10%-points, the data will be considered supportive of the non-
inferiority hypothesis. 
 
The 10% non-inferiority margin has been obtained by a DELPHI method talking to many 
infectious disease specialists – and also used in the WHO coordinated ABCD trial. The 
margin is expressed as an absolute and not a relative difference in AMR prevalence, 
because absolute increase in AMR prevalence will indicate how many additional possible 
exposures with resistant bacteria the study area population may face. Thus, an increase 
from 1% to 11% or 50% to 60% will provide 10 new “risk” persons in a group of 
hundred even if one goes up in relative terms by 1000% and the other only 20%. 
 
We will retain and freeze at -80oC the original swabs after plating as well as the identified 
bacterial colonies, to allow further analyses with the indicator bacteria or any other 
intestinal bacteria or other microbes with later evolving methods. Bacterial colonies will 
be stored at two points for later analyses: during primary culture when organisms other 
than the main targets are grown and from the antibiotic sensitivity plate in order to 
capture resistance diversity within the target organisms. The stored cultures can 
subsequently be analysed through standard culture or by molecular techniques.  
 
Figure 6 summarises samples taken for AMR analyses and stored at -80oC. In total, there 
will be an estimated of 54,000 vials, without the so called “clinical isolates”, taken from 
ill children. In total, these stored samples thus will fill one -80oC freezer. 

Trial protocol: LAKANA 
Page 35 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
 
12-month post 
intervention 
15-26 
mo 
0 
Nasopharyngeal 
swap 
STGG media 
2 
100ug 
-80o 
1000 
12-month post 
intervention 
15-26 
mo 
0 
Rectal swab 
Cairy-Blair media 
1 
100ug 
-80o 
1000 
12-month post 
intervention 
15-26 
mo 
0 
Rectal swab 
DESS Media 
1 
100ug 
-80o 
1000 
12-month post 
intervention 
15-26 
mo 
0 
Rectal swab 
None, dry swab 
1 
100ug 
-80o 
1000 
12-month post 
treatment (in 
villages with 
MDA) 
24-35 
mo 
0 
Nasopharyngeal 
swap 
STGG media 
2 
100ug 
-80o 
600 
12-month post 
treatment (in 
villages with 
MDA) 
24-35 
mo 
0 
Rectal swab 
Cairy-Blair media 
1 
100ug 
-80o 
600 
12-month post 
treatment (in 
villages with 
MDA) 
24-35 
mo 
0 
Rectal swab 
DESS Media 
1 
100ug 
-80o 
600 
12-month post 
treatment (in 
villages with 
MDA) 
24-35 
mo 
0 
Rectal swab 
None, dry swab 
1 
100ug 
-80o 
600 
 
 
To gain a standardized understanding of antibiotics utilized for common clinical 
syndromes in the study area, we will collect and analyse national and regional guidelines 
and protocols on antibiotic prescribing on an annual basis. We also aim to monitor the 
amount of common antibiotics sent out to health facilities from the six study districts. A 
Duration of 
intervention in 
community
Child age
# of given 
MDA rounds 
/ village
Biological 
specimen
Storage media
Estimated # 
of aliquots
Volume / 
aliquot
Storage 
temperature # of children
0 months
4-14 mo & 
49-59 mo
0
Nasopharyngeal 
swab
STGG media
2
100 ug
-80o
1350 + 1350
0 months
4-14 mo & 
49-59 mo
0
Rectal swab
Cary-Blair media
1
100 ug
-80o
1350 + 1350
0 months
4-14 mo & 
49-59 mo
0
Rectal swab
DESS Media
1
100 ug
-80o
1350 + 1350
0 months
4-14 mo & 
49-59 mo
0
Rectal swab
None, stored dry
1
100 ug
-80o
1350 + 1350
12 months
4-14 mo & 
49-59 mo
2
Nasopharyngeal 
swab
STGG media
2
100 ug
-80o
1350 + 1350
12 months
4-14 mo & 
49-59 mo
2
Rectal swab
Cary-Blair media
1
100 ug
-80o
1350 + 1350
12 months
4-14 mo & 
49-59 mo
2
Rectal swab
DESS Media
1
100 ug
-80o
1350 + 1350
12 months
4-14 mo & 
49-59 mo
2
Rectal swab
None, stored dry
1
100 ug
-80o
1350 + 1350
24 months
4-14 mo & 
49-59 mo
6
Nasopharyngeal 
swab
STGG media
2
100 ug
-80o
1350 + 1350
24 months
4-14 mo & 
49-59 mo
6
Rectal swab
Cary-Blair media
1
100 ug
-80o
1350 + 1350
24 months
4-14 mo & 
49-59 mo
6
Rectal swab
DESS Media
1
100 ug
-80o
1350 + 1350
24 months
4-14 mo & 
49-59 mo
6
Rectal swab
None, stored dry
1
100 ug
-80o
1350 + 1350
12 months post-
intervention
4-14 mo & 
49-59 mo
0
Nasopharyngeal 
swab
STGG media
2
100 ug
-80o
1350 + 1350
12 months post-
intervention
4-14 mo & 
49-59 mo
0
Rectal swab
Cary-Blair media
1
100 ug
-80o
1350 + 1350
12 months post-
intervention
4-14 mo & 
49-59 mo
0
Rectal swab
DESS Media
1
100 ug
-80o
1350 + 1350
12 months post-
intervention
4-14 mo & 
49-59 mo
0
Rectal swab
None, stored dry
1
100 ug
-80o
1350 + 1350

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
more detailed review of antibiotic prescription will be done in the four health facilities in 
which the AMR sample collection takes place.  
 
4.17. Data collection on mechanisms of action for azithromycin 
 
Azithromycin MDA has been associated with reduced child mortality, but its mechanism 
of action in this respect remains unclear. The drug is a broad-spectrum antibiotic with 
strong activity against many bacterial species and it also has anti-inflammatory properties 
(Kwiatkowska and Maślińska, 2012). A recent report from the MORDOR trial in Niger 
documented a lower population prevalence of malaria in communities where under-five-
year-old children had received azithromycin than in control villages, but it is not known if 
this was caused by a direct antimalarial effect or through an indirect pathway (Arzika et 
al., 2019). Antibiotic treatment might also affect infants’ immunity through multiple 
pathways, including alterations in enteric microbiota and pathogens, enteric and general 
inflammation, and immune development.  
 
The current trial provides an excellent opportunity for a sub-study to test hypotheses 
about mechanisms of azithromycin activity against child mortality. In LAKANA, we will 
test three hypotheses that have been posed but not tested directly earlier, i.e. that 
azithromycin MDA eliminates malaria parasitaemia and reduces inflammation in the 
treated host. We will also store biological samples for later laboratory analyses on other 
hypotheses, to be more specifically determined once the mortality effect is known and 
some ongoing mechanistic studies elsewhere have been completed. 
 
These are the three hypotheses that we will test in the first phase: 
 
1. Biannual or quarterly azithromycin MDA to 1-11 month (29-364 days) old infants 
eliminates their asymptomatic malaria, reduces population prevalence of malaria 
parasitemia, and improves the infants’ mean blood hemoglobin concentration.  
a. Malaria control may be a factor influencing mortality. Azithromycin effect 
on malaria can in the LAKANA trial be assessed both with direct 
parasitemia analysis among 4–11-month-old children before and 2 weeks 
after the fourth azithromycin or placebo treatment and by comparing 
prevalence of malaria parasitemia among 6–8-month-old and 12–14-
month-old children who live in the study villages and who have been 
eligible to receive 2-4 doses of the study drug. As a marker of health 
consequences of malaria treatment, we can additionally study blood 
hemoglobin concentration in the same children. 
b. To assess community-level prevalence of malaria across communities at 
the end of the intervention, and a possible carry-over effect, we will also 
study prevalence of malaria among 1-59-month-old children. This survey 
will be conducted on visit 9, i.e., when the intervention has lasted for 2 
years. The sample size for this survey will be 3000 children aged 1-59 
months. The Probability-proportional-to-size (PPS) sampling method will 
allow that each household in the sample has the same probability of being 
selected. In each household selected, all 1-59-month-old children will be 
tested with a malaria rapid diagnostic test.   

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
2. Biannual or quarterly azithromycin MDA to 1-11 month (29-364 days) old infants 
reduces their general inflammation.  
a. General inflammation may be an important driver of subsequent disease 
acquisition and activity. This may be driven by bacteria resident on 
mucosal surfaces or through the inflammatory modulating properties of 
azithromycin and can be reflected using simple measure of CRP in small 
volumes of plasma, in the same groups of children from whom we will do 
the malaria analyses.  
b. Larger plasma samples and peripheral blood mononuclear cells from 6–8-
month-old and 12–14-month-old children who have been eligible to 
receive 2-4 doses of study drug will be used for more extensive 
inflammatory profiling as previously described in our studies on immune 
activation, inflammation and antibiotic impact in children with HIV 
(Fitzgerald et al., 2016; Prendergast et al., 2016; Bourke at al., 2019). We 
will focus on markers we have found to distinguish children with evidence 
of increased inflammation and immune activation which include, IL6, IL8, 
IL10, TNF, IL1-RA, soluble adhesion molecules, Serum Amyloid A, 
markers of disordered thrombogenesis, and markers of cell activation and 
proliferation (HLA-DR, CD38, Ki67) on naïve and memory CD4 and CD8 
populations. 
 
3. Biannual or quarterly azithromycin MDA to 1-11 month (29-364 days) old infants 
reduces their intestinal inflammation. 
a. In addition to any impact on general inflammation, intestinal inflammation 
and environmental enteric dysfunction (EED) could be an important driver 
of disease. This will be studied by measuring neopterin, myeloperoxidase, 
and alpha-1-antitrypsin (and possibly other biomarker) concentrations in 
stools collected from 4–11-month-old infants before and two weeks after 
the fourth MDA and among 6–8-month-old and 12–14-month-old children 
who live in the study villages and who have been eligible to receive 2-4 
doses of the study drug. 
 
After the trial, we propose to test the following hypotheses (and possibly others) if they 
are still valid then. For LAKANA, the budget includes only sample collection and storage 
for these analyses, funding for the actual analyses will be sought later. 
 
4. Biannual or quarterly azithromycin MDA to 1-11 month (29-364 days) old infants 
leads to improved immunity and vaccine response among 6–14-month-old 
children. 
a. Vaccine efficacy is reduced in low-income settings, potentially due to 
heavy microbial load (Parker et al., 2018). In some settings, it has been 
possible to improve vaccine response by sanitation and hygiene 
interventions (Church et at, 2019) or antimicrobials given before 
vaccination (Uchiyama et al., 2014). This suggests vaccine response 
enhancement as one of potential mechanisms by which azithromycin could 
reduce infant mortality. As markers of vaccine response, we will in the 
LAKANA trial measure antibody titers to rotavirus, tetanus, and measles 
vaccines. Rotaviral and tetanus antibody concentrations will be analyzed 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
from plasma samples collected at 6-8 months of age and measles antibody 
concentration at 12-14 months of age. 
b. Azithromycin could also modulate cellular immunity, either directly or 
indirectly, as we have previously described for the antibiotic 
cotrimoxazole (Bourke et al., 2019). We and others have shown that the 
ratio of naïve-to-memory CD4+ and CD8+ T-cells was much lower in 
African children than in resource-rich settings. Our data shows that this is 
probably driven by infection driving T cell activation and memory cell 
expansion despite greater thymic output (Klein et al., manuscript in 
preparation). In LAKANA, we will determine if azithromycin modulates 
thymic output, the ratio of naïve to memory CD4 and CD8 sub-
populations, their cytokine production and proliferation, and T cell 
receptor repertoires (TCR) (Gibbons et al., 2014; Sandgaard et al., 2014; 
Gkazi et al., 2018).  
 
5. Biannual or quarterly azithromycin MDA to 1-11 month (29-364 days) old infants 
eliminates specific pathogenic microbes from their intestinal tract.  
a. Elimination of specific pathogenic microbes is one of the key hypotheses 
on how azithromycin might reduce child mortality. In LAKANA, this 
hypothesis can be tested using TaqMan cards of 40-50 potentially 
pathogenic intestinal microbes and stored stool samples from the trial 
participants. The analyses can be done from paired stool samples collected 
from 4–11-month-old infants before and two weeks after the fourth MDA 
(to track what happens with one MDA dose on an individual level) and 
from 6–8-month-old and 12–14-month-old children who live in the study 
villages and who have been eligible to receive 2-4 doses of the study drug 
(to see a cumulative effect of multiple doses). 
 
6. Biannual or quarterly azithromycin MDA to 1-11 month (29-364 days) old infants 
modulates their critical bacterially driven metabolic pathways. 
a. Metabolic profiling in combination with fecal microbiome will be 
performed in stored plasma, urine and stool as previously described 
(Wijeyesekera et al., 2019). Fecal and urine metabolites will be 
determined by 1H nuclear magnetic resonance (NMR) spectroscopy. The 
extent to which the metabolic signature obtained reflects the 
corresponding microbiota state will be possible combining microbiome 
data. Special attention will be given to the presence of short-chain fatty 
acids (acetate/propionate/butyrate/valerate and their isoforms) as well as 
studying bile-acid metabolism, which play a pivotal role in many aspects 
of microbial ecology, most importantly in maintaining community 
homeostasis; their potential contribution to ‘mono-domination’ and 
‘stochastic dysbiosis’ following azithromycin administration.  
 
7. Biannual or quarterly azithromycin MDA to 1-11 month (29-364 days) old infants 
does not cause a sustained reduction in the diversity and immune impact of their 
intestinal microbiota (measured at one year after the last provided MDA).  
a. The composition of the microbiota is known to be influenced by infections 
and antibiotic usage. Modelling these changes over time is typically 
challenging due to the large number of potential interactions between 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
species. However, by using diversity relative to baseline as a measure of 
displacement from an ‘equilibrium state’, our group has recently 
developed a simple analytical model of microbiota reconstitution after 
antibiotic treatment (Shaw et al., 2019). This model successfully shows 
that short courses of common antibiotics (including ciprofloxacin, 
clindamycin, minocycline, and amoxicillin) can have year-long effects in 
adults, causing transitions to alternative microbiota states (quantified using 
diversity relative to baseline). We will employ this modelling approach to 
quantify the in vivo impact of antibiotics on our patients. By fitting the 
model to different patient characteristics (e.g., age, weight, MDA) we will 
also be able to compare model parameters and comment on the potentially 
different impacts of antibiotics between groups. Simultaneously 
quantifying microbiota stability and immunity over time, we will be able 
to study how the two interact and their relative contribution to clinical 
outcomes using multivariable time series modelling. 
 
Data collection for these analyses will be done in the same sub-sample of 60 clusters as 
used for the AMR work and other secondary outcomes. From these clusters, we will 
invite three groups of children for the mechanistic studies: 
1. 4–11-month-old infants living in communities that will receive their 4th MDA.  
2. 12–14-month-old infants who live in communities that will receive their 6th, 7h, 
8th study MDA and at 9th study visit.  
3. 6–8-month-old infants who live in communities that will receive 6th, 7th, 8th study 
MDA and at 9th study visit. 
 
We will test the first three of the above-described hypotheses (about malaria 
elimination and reduction of general or intestinal inflammation) from the first group 
of children, i.e., 4–11-month-old infants living in communities receiving their 4th 
MDA. From these infants, a study nurse will collect a heel-prick blood sample and a 
flocculated rectal swab before they have received any MDA. They will collect 
another heel prick blood sample and a stool sample (or rectal swab if stool collection 
fails) two weeks after the 4th round of MDA has been conducted in their 
communities. From the collected blood, the nurse will measure hemoglobin and CRP 
concentration with an on-site reader and store blood on a filter paper for later malaria 
diagnostics with DNA-amplification. From the stool sample, a laboratory technician 
will determine calprotectin and alpha-1-antitrypsin concentrations with commercially 
available ELISA tests. Additional stool and urine samples will be collected and stored 
for later analyses (on hypotheses 5-7). In the analysis phase, we will compare 
prevalence of malaria parasitemia, mean blood hemoglobin and CRP concentration 
and mean fecal neopterin, myeloperoxidase, and alpha-1-antitrypsin concentration in 
the three study arms, both before and after the first MDA treatment. 
 
From the second and third group of children (12–14-month-olds who have received 3-
4 doses of MDA and 6–8-month-olds who have received 2-3 doses of MDA) we will 
test hypotheses 2-4, i.e., those about broader general and intestinal inflammation and 
immune function and development. These infants will be invited to a health facility or 
a central location in a village - after the MDA visit to their household. At the clinic, a 
study nurse will receive a home collected stool sample or take a rectal swab if stool 
collection has failed. S/he will also draw a 5 ml blood sample and collect a 10 ml 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
urine sample, which will be delivered to a nearby study laboratory by a motorcycle 
messenger. A laboratory technician will measure Hb, separate plasma and white 
blood cells, aliquot plasma, urine and stool, and store the samples at -80oC freezer. 
Approximately once a month, the stored samples will be shipped to a central 
laboratory at CVD-Mali in Bamako, where another laboratory technician will perform 
the immunological and inflammatory assays. In the analysis phase, we will compare 
mean plasma concentrations of vaccine antibodies and selected cytokines, thymic 
output, the ratio of naïve to memory CD4 and CD8 sub-populations in the three study 
arms. For the azi-biannual group, we will perform an additional exploratory analysis 
to assess the impact of timing of the azithromycin dose (given at 1-2 and 6-8 months 
of age vs at 3-5 and 9-11 months of age). 
 
For hypothesis on microbial clearance and reduction of inflammation, we expect to 
see the largest effect in infants who have not received azithromycin earlier. We also 
expect that age of the child may be an effect modifier. Hence, we primarily focus on 
one time-point (fourth MDA) and a wide age-bracket (4–11-month-old infants). The 
youngest infants (less than 4 months old) will be excluded for logistical reasons as 
they will not be invited to sample collection for AMR analyses. Since we want to be 
able to track individual level changes in test variables, we will collect and analyse 
samples before and after the MDA. The chosen blood analyses can be completed a 
battery-operated on-site reader from a small volume taken with a heel-prick – and 
hence we will not need to take any venous samples from these children. 
 
For the hypotheses on immune function and development, we expect to see the largest 
effect among infants who have received repeated MDA doses. Therefore, we will 
choose children at a time when there have been at least 4 rounds of MDA in their 
home villages. At this point, most or all the 12–14-month-old children have received 
3-4 MDA doses and 6–8-month-old children have received 2-3 doses. For the 
immune function and maturity analyses, we will need a larger blood volume and 
hence we will take it by a venous puncture, at a study clinic after MDA. Since we are 
interested in a cumulative effect of multiple MDA doses and only want to do group 
level comparisons, there is no need for paired sample collection. 
 
The age groups have been selected in a way that there will be no more than one 
venous blood sample per child during the study. From the 6-8-month of children, we 
will primarily assess immune development and vaccine responses to tetanus and 
rotaviral vaccines (given before the age of 6 months). From the 12-14-month of 
children, we will assess immune development and vaccine response to measles 
vaccine (given usually between 9 and 12 months of age). From both age-groups, we 
will also assess malaria prevalence by a DNA amplification test. Immune 
development and infection are best studied among over-6-month-old children, since 
by this age infants will have lost most of their passive maternal immunity and will be 
increasingly susceptible for infection, inflammation, dysbiosis, and EED. 
 
Figure 7 summarises analyses planned to be completed in the first phase.  
 

Trial protocol: LAKANA 
Page 41 / 102 
 
Protocol version 7.0: 13. November. 2024 
 
 
 
Figure 8 summarises biological samples planned to be stored from the study participants 
(in addition to those, stored in the AMR part of the study). These samples will be used 
address hypotheses 5-7 of the mechanistic studies as well as for any additional 
hypotheses formulated during the trial. If all samples were collected and aliquoted as 
planned, there would be a total of 51,800 stored vials, i.e., these samples will fill one -
80oC freezer. 
 
 
 
As shown in the figure, we estimate to collect samples from approximately 1,000 infants 
at the fourth MDA visit and 1,350 thereafter, i.e., approx. 330 - 450 / intervention group.  
 
 
4.18. Stakeholder perspectives on feasibility, including economic analyses 
 
As previously stated in this protocol (p. 11), in the case of positive clinical results from 
the LAKANA trial, there is an intention (although not necessarily yet a political 
commitment) to implement a routine national public health intervention for azithromycin 
Biological 
specimen
Child age
MDA or 
study 
visit
Timing of lab 
sample vs MDA
# of given 
MDA doses
Storage media
Estimated # 
of aliquots
Volume / 
aliquot
Storage 
temperature # of children
Blood
4-11 mo
4
Before
1-3
Filter paper
2
50 ul
-80o
1000
Stool
4-11 mo
4
Before
1-3
Cryovial
4
1.5 g
-80o
1000
Urine
4-11 mo
4
Before
1-3
Cryovial
3
1.5 ml
-80o
1000
Blood
4-11 mo
4
 14 d after
1-3
Filter paper
2
50 ul
-80o
1000
Stool
4-11 mo
4
 14 d after
1-3
Cryovial
4
1.5 g
-80o
1000
Urine
4-11 mo
4
 14 d after
1-3
Cryovial
3
1.5 ml
-80o
1000
Plasma
6-8 mo
6-9
Before
2-3
Cryovial
4
500 ul
-80o
1350
Peripheral blood 
mononuclear cells
6-8 mo
6-9
Before
2-3
Cryovial
3
500 ul
-80o
1350
Stool
6-8 mo
6-9
Before
2-3
Cryovial
4
1.5 g
-80o
1350
Urine
6-8 mo
6-9
Before
2-3
Cryovial
3
1.5 ml
-80o
1350
Plasma
12-14 mo
6-9
Before
3-4
Cryovial
4
500 ul
-80o
1350
Peripheral blood 
mononuclear cells 12-14 mo
6-9
Before
3-4
Cryovial
3
500 ul
-80o
1350
Stool
12-14 mo
6-9
Before
3-4
Cryovial
4
1.5 g
-80o
1350
Urine
12-14 mo
6-9
Before
3-4
Cryovial
3
1.5 ml
-80o
1350

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
administration in Mali. By studying demand and supply side determinants, the feasibility 
sub-study aims at providing comprehensive policy advice to the decision makers on if 
and how this intervention could be implemented, what can be the bottlenecks and how to 
go around them.  
 
The feasibility study draws from data collected during the main LAKANA clinical trial, 
but it will also collect data which is not directly linked to the clinical trial MDA approach 
in order to explore delivery options which are beyond implementation of the current 
clinical trial protocol. Going beyond the clinical trial protocol is a necessity as any 
routine public health intervention model would not have the same type of research 
component as a clinical trial. 
 
The feasibility study framework breaks down to four research components:  
1. 
Acceptability  
2. 
Equity  
3. 
Health system compatibility  
4. 
Economic analysis based on costing and cost-effectiveness analysis  
 
Figure 9 below depicts framework for the feasibility study and its components. 
 
 
 
 
 
Acceptability 
Understanding perspectives and views of various stakeholders on the current LAKANA 
MDA intervention will form the basis of the acceptability study component. The aim of 
the acceptability study is to describe how stakeholders conceptualize, perceive and 
experience the current LAKANA MDA intervention and how they might conceptualize 
and perceive a future national routine intervention.  
 
Acceptability is a multi-faceted construct that reflects the extent to which stakeholders 
consider an Azithromycin intervention to be appropriate, based on anticipated or 
experienced cognitive and emotional responses to the intervention (Sekhon at al 2017). 
We will confine the present study on health system and household levels (those 
delivering and receiving the intervention) because of the important role they play. We 
will focus on local practices and interpretations related to child health, illnesses and well-
being and will provide a mostly qualitative analysis of the acceptability of the drug and 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
the delivery model. 
 
The acceptability study stream will rely on a mixed methods approach: for collecting 
qualitative data, we will conduct focus-group discussions and semi-structured interviews 
with families, health workers, relais, village chiefs and elders and other key stakeholder 
groups. Interviews will take place during the trial implementation. Additionally, 
throughout the intervention, we will conduct in-depth interviews with other stakeholders 
such as health sector decision makers and politicians. The number of qualitative 
interviews needed will be based on data saturation. Typically, the total number would be 
around 50 interviews. We will contextualize the qualitative data using quantitative data 
collected from all participating households (household socio-economic status, location of 
residence, family composition and other background variables).  
We will also collect quantitative data from households through a questionnaire which will 
be administered in the 59 sub-study villages. The questionnaires will cover questions 
related to acceptability but also to the economic analysis (see below)  
 
The CVD social science team in collaboration with Point Sud research unit will conduct 
qualitative interviews in the vernacular (Bambara), transcribed and translated into French. 
The CVD social science team, together with Tampere University feasibility team will 
read the interviews immediately after they have been conducted and add new themes to 
cover topics that remain unanswered to the interview guide. The analysis of the 
qualitative data will be done using a multipronged approach including thematic and 
discourse analysis. The analysis might include use of a software such as Atlas.ti. We will 
analyse quantitative data for proportions and means using Stata software. 
 
Equity 
The drugs will be distributed in a research context aiming to determine efficacy of 
azithromycin. By collecting various types of background information from the study 
participants and assessing if the background variables (such as poverty or household 
location), we will be able to assess the equity of impact of this intervention. However, 
since there will be no data on those whom the study team does not reach, villages that 
refuse participation, or areas that are excluded from the study due to security or other 
issues, we will not be able to quantitatively measure equity of access to the intervention. 
Instead, we will assess equity of access indirectly, using qualitative interviews among 
stakeholder groups. We will use snowball sampling for selecting the interviewees, but 
preliminary, we plan to include representatives of multilateral organizations, funding 
partners, national Ministry of Health (MOH) teams, district MOH teams, volunteer rural 
health workers, and community members participating in MDA campaigns. In this sub-
study, we aim to explore the possible bottlenecks of achieving effective coverage and 
ways to overcome the difficulties in implementation. 
 
We will consider the village location and accessibility as an equital aspect: we will treat 
the village’s Strategie Avancée status as a potential effect modifier of azithromycin 
MDA. Additionally, we will conduct a survey amongst the field workers to obtain a 
subjective assessment on how difficult the villages are to reach as a health worker. The 
results will be assessed in the context of whether the district or health area has a system in 
place to take the remote and difficult locations into account in terms of arranging health 
care.  
 

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Protocol version 7.0: 13. November. 2024 
 
 
Health system compatibility 
The health system compatibility study component aims at answering the study question 
of: what are the health system compatibility parameters for different implementation 
scenarios for an Azithromycin public health intervention in the context of Mali?  
 
This study stream will include questions on the transferability of the LAKANA 
intervention to a scaled-up national intervention, but it will go beyond the LAKANA trial 
implementation by exploring other possible implementation models. The different 
possible implementation models are assessed through supply side elements (health 
system building blocks) in the Malian context. The implementation models will detail the 
people and institutions involved in each model and the process of implementation of each 
model. 
 
The health system compatibility study stream will be based on literature and observations 
on the Malian health system and on interviews with key stakeholders, such as health 
system researchers, health policy makers, health workers at different levels of the system. 
The health system compatibility study stream will also draw from lessons learned in 
implementing the LAKANA trial.  
 
The theoretical framework for the health system compatibility study stream relies on 
implementation research / science. Bauer et al (2015) have defined implementation 
science as “The study of methods to promote the adoption and integration of evidence-
based practices, policies, research findings and evidence into healthcare policy and 
practice.” 
 
Economic analysis based on costing and cost-effectiveness analysis 
The economic analysis study component aims at answering the study question of: what is 
the cost of the different implementation models and what are the differences in cost-
effectiveness between the different models? The costing exercise will be based on an 
ingredients approach in which all the different inputs or ingredients that are needed to 
produce one unit of the intervention need to be costed trough market prices or prices 
derived from competitive market prices. All the prices will be translated into one single 
monetary unit.   
 
The costs include in the costing are direct, indirect and opportunity costs. The direct costs 
will be mostly derived and modelled from the LAKANA trial costs with the deduction of 
research costs. These costs will be modelled to represent the base line MDA approach. 
For the other implementation models the costing model will require further adjustments 
according to how the models will be defined. This adjustment can only be done after the 
implementation models have been developed.   
 
The costing exercise will also include household level opportunity cost. This equates to 
the indirect cost paid by the participants, more precisely the care givers bringing their 
children to receive the Azithromycin intervention. To calculate the household level cost 
the intervention logic (as described above) will need to cover also the household 
perspective, i.e., what is required from the household (in terms of time and indirect 
payments to reach the point of service) to receive the service. 
 

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Protocol version 7.0: 13. November. 2024 
 
The effectiveness will be determined by the trial outcome – both the primary outcome of 
mortality and the secondary outcomes of morbidity and possible AMR effects. The 
effectiveness can be expressed in different units such as deaths averted, or disability 
adjusted life years (DALYs).  
 
 
Theoretical framework for the costing and cost-effectiveness analysis are based on 
standard economic evaluation methods – with focus on theoretical frameworks covering 
economic evaluation of public health programmes (WHO 2004; NICE International, 
2014; Sohn et al., 2020). 
 
 
4.19. Strategie Avancée and field workers’ assessment of hard-to-reach areas  
 
Villages under Strategie Avancée will be identified by the local field supervisors from the 
records maintained by the local districts and health areas. The Stategie Avancée status 
information will be appended to the study data prior to the analysis by the study data 
manager. 
 
We will conduct a survey amongst the trial data collection staff to obtain a subjective 
assessment on what is the geographical accessibility of a village. The overarching aims of 
the survey for the data collectors would be to develop an instrument to assess the 
hardness-to-reach from the point of view of data collectors or health care workers going 
to the locations, and to evaluate whether hardness-to-reach modify the effectiveness of 
LAKANA intervention. The survey will be implemented as an electronic questionnaire 
and the survey will be anonymous: no staff name or position will be recorded. 
 
The participants will be approached by their field supervisors with an announcement of 
an invitation to fill the questionnaire based on their own experiences. Filling the 
questionnaire will be voluntary, but they will be informed on the benefits of what such 
information can provide: insight on their challenges during the data collection, an 
improved viewpoint on the collected trial data with hardness-to-reach as a predictor or 
effect modifier on the trial outcomes, and information that could improve the planning of 
future interventions that could help even the most remote communities. 
 
The participants will be asked to fill the questionnaire before synchronizing the tablets for 
data collection, either at the beginning of the day or at the end of the day, at their 
convenience. The questions will be in French language, and at the beginning of the 
questionnaire, there will a brief introduction and instructions on filling the electric form.  
 
The form will consist of 12 questions for a single village. Each field worker will fill the 
form for 10 villages. The questions will be multiple choice questions and filling the form 
for one village will take approximately 5 minutes.  
 
The expected number of answers will be 75% of the available field workers. The field 
supervisors will be reminded to ask the field workers to fill the form until 75% is reached, 
or until the field supervisors report that there are no more field workers who are willing 
to fill the form. As the data collectors enter villages in teams, there are multiple data 

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Protocol version 7.0: 13. November. 2024 
 
collectors who have experience from a single village. Each village should be assessed by 
the full team of field workers, that is, each village should have at minimum two 
assessment by separate field workers. 
 
There will be multiple survey rounds: the survey will be tested with 5 data collectors to 
assess the workload filling the forms for multiple villages, and to assess the data quality. 
If the data quality suffers from the workload of filling the questionnaire for many 
villages, the length of the electronic data capture form will be reduced by allowing the 
field workers to continue form filling at the later time. 
 
 
4.20. Data collection on trial safety 
 
We will monitor intervention safety in LAKANA with two approaches: 
 
1. Passive surveillance for the incidence of serious adverse events (SAE) within 14 
days of study drug administration, in the whole study area 
2. Active surveillance for the incidence of SAEs and adverse events (AE) within 14 
days of study drug administration, in the 60 villages selected for more detailed 
data collection on outcomes other than mortality. 
 
The passive surveillance will be done through health facilities in the study area. Whilst an 
MDA round is being implemented in any location and and for two weeks thereafter, a 
study coordinator will visit or call the local health center on each week-day and record 
any suspected SAEs that have been notified among 1-11 month old infants at the facility. 
Caregivers of infants who receive study drugs are also adviced to notify study team 
members if the infant is hospitalised within two weeks of the MDA.  
 
The following events will be considered SAEs, if they occur within 14 days of study drug 
administration: Death, life-threatening event, hospitalization, and other serious events as 
judged by a study physician.  
 
Active surveillance for SAE and AE will be completed for participants in the 60 clusters 
selected for more detailed data collection on a number of secondary outcomes and other 
variables. Infants aged 4-11 month old will be invited to the health facilities or central 
location in a village two weeks after the 4th round of MDA has been conducted in their 
communities. At this visit, a study nurse or another trained study team member will also 
interview a caregiver about symptoms that the infant has experienced after the 
azithromycin MDA and that may possibly be related to it. 
 
 
4.21. Group allocation and trial code managament 
 
Participating study clusters (villages) will be randomly allocated into the three 
intervention groups at 3:2:4 ratio (control: azi-quarterly: azi-biannual), in public 
allocation events where village representatives blindly pull lottery tickets out of a 
container. The randomization will be stratified by cluster size (below or above 100 

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infants / cluster) and by district, i.e., each participating district will have its own 
randomization event. The randomization lists (trial code) will be stored electronically, in 
a password protected file, by a statistician not involved in trial implementation, the data 
management consulting company, and by the Chairman of the trial’s data safety and 
monitoring board (DSMB). 
 
The trial code will be opened when all trial data for the 24-month follow-up visit have 
been entered into a computer database and data accuracy has been verified. To facilitate 
an analysis on mortality and other efficacy outcomes whilst follow-up for AMR is still 
going on, a statistician will temporarily break the code, add group information to a 
database containing the efficacy outcomes and then recode participant and cluster codes 
into new identifiers that cannot be linked to the actual identification. With this approach, 
the statistician can then share a full database with researchers analysing the efficacy 
outcomes, whilst others continuing the data collection of AMR outcomes will remain 
blinded to the group code. 
 
4.22. Methods for protecting against other sources of bias 
 
Randomisation into the trial, group allocation, and distribution of study drugs will be 
done by personnel not participating in the outcome evaluation.  
 
The interventions will be double-masked, i.e., the study drugs will look and taste identical 
and coded only with a letter code.  
 
To ensure adherence, all study drugs are given under direct observation. 
 
 
 
 

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4.23. Withdrawal from participation after enrollment 
 
The enrolled household representatives can decide to discontinue the study participation 
or decline provision of study drug to an infant at any point. If known, the reason for 
household withdrawal or study drug denial will be indicated in electronic Case Report 
Forms. The participants can, however, withdraw their consent or decline study provision 
to a child without giving any reason for their withdrawal. 
 
Individuals experiencing a serious adverse event that is likely to be related to the trial 
intervention are withdrawn from further study drug provision by the investigators (but 
will be included in the follow-up and analysis).  
 
Withdrawn or discontinuing participants will not be replaced by others, i.e. possible 
dropouts will not influence the sample size, once the study has started. The study team 
will compare the baseline characteristics of discontinuing households to those who do not 
discontinue, in order to assess the possibility of follow-up bias. 
 
 
4.24. Other treatments to trial participants 
 
During and after the trial, members of all participating households (i.e., those completing 
the planned follow-up period as well as those withdrawing at some point) will receive 
standard preventive and curative health services, as recommended and provided by the 
national health system. SMC with amodiaquine and sulfadoxine-pyrimethamine will be 
offered through the national health service to all 3–59-month-old children on a monthly 
basis between July and October-November in each year. 
 
At the end of the trial, if the analysis suggests a mortality reduction by azithromycin, all 
1-11 month (29-364 days) old infants in the participating households will be offered a 20 
mg / kg dose of azithromycin. If deemed appropriate thereafter, the study team will work 
with Malian Ministry of Health to roll-out a longer-term azithromycin MDA program at 
the trial site and elsewhere in Mali. 
 
 
4.25. Co-enrollment guidelines 
 
LAKANA trial participants are encouraged not to enrol in other clinical trials using 
antibiotics before the end of the LAKANA follow-up period. However, participation in or 
enrollment to another trial is not an exclusion criterion, i.e. child can be included in the 
trial and receive study drug MDA if s/he was participating also in another trial. 
 
 
4.26. Storage of biological samples 
 
All collected biological samples will originally be shipped daily to study laboratory in 
Kita, where they will be processed and stored at a project freezer (-80oC). On a monthly 

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basis, the samples will be shipped to a main laboratory at CVD-Mali in Bamako and 
stored at -80oC. All biological samples will be aliquoted in small volumes, to allow 
appropriate vials to be easily shipped to various collaborators, where they will be stored 
until analysis. Chapters 4.15 and 4.16 gives details of the stored aliquots. 
 
 
4.27. Data recording and management 
 
All data will be captured electronically, using a custom-made data entry software 
installed on personal smartphones or small tablet computers. The exact software is yet to 
be decided but the data management approach will involve offline data collection, mostly 
at participant’s home. Data transmission to a cloud-based server will occur in real-time 
from households in the field or daily from cluster centers, using a 3G broadband 
connection. All server-based data will be backed up to another location daily. 
 
Access to the tablet computers and the cloud-based data will be username and password 
protected. During transmission and at the cloud, data will be encrypted to ensure data 
privacy. The tablet computers will be stored and charged overnight at locked study 
offices that will have safety and electricity. 
 
Error and inconsistency checks will be automated in the software. A data manager will 
review new data entries and full data report on a weekly basis (daily at the beginning of 
data collection) and make corrections where appropriate, to ensure data integrity.  
 
All documentation regarding the participants, including the laboratory samples, source 
data and the Case Report Forms, will be identified in the database with appropriate 
participant codes. The names will only appear on informed consent forms and a separate 
coding list. A minimal set of identifying information, necessary for the implementation of 
the trial, will be stored in an encrypted form on the data collection devises and in a 
separate encrypted database. Access to the identifying information will be limited to 
authorised data collectors with a secure username and password.  
 
The investigators shall maintain electronic records of study drug receipts and transfer 
logs and electronic copies of the Case Report Forms and regulatory documents (informed 
consents, ethical approval) for 5 years after the end of the study, as advised by local 
authorities. All records will be kept in a secure place. Clinical information will not be 
released without written permission of the subject, except as necessary for monitoring. 
 
Selected data (participant’s name, sex, date of birth, date of last visit, home village, 
household identification number, enrollment date and enrollment number) will be 
summarised in an electronic register. The registers are stored in encrypted form and 
accessible only with an authorised username and password in the data collectors tablet 
computers (to facilitate participant identification) and a cloud-based folder. Data from 
this register may be given to selected members of the research teams and authorities 
guiding health research in Mali or Finland. 
 
After the trial completion, the study team will place the study database and respective 
metadata publicly available, at a suitable non-profit internet-site (such as ClinEpiDB). 

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The study team will contract an external partner (RTI International) for the development 
of the data entry and management system support for its maintenance. After the initial 14 
months, i.e. when the system is running and enrollment almost completed, the study team 
will assume full responsibility of data management. 
 
 
4.28. Pilot study 
 
There will be a 3-month pilot phase, during which the team will hire and train study staff 
and test standard operating procedures, data collection forms, and data management. 
Many practical arrangements and standard operating procedures (SOPs) will be adopted 
from earlier ABCD and SANTE -trials, also testing various aspects of azithromycin 
impacts on children and currently being carried out in Mali, with the same Malian co-PI 
as in LAKANA trial. 
 
Mortality data from the first two 3-month intervals, from the villages that will be enrolled 
in the first week of trial activities, will be treated as pilot data and not included in the 
main database for trial analysis. 
 
 
4.29. Quality assurance and control 
 
Internal quality assurance is implemented using standard operating procedures (SOPs) 
and weekly internal monitoring. The team in Mali will employ 1-3 quality assurance 
officers, who will regularly work with the staff on quality assurance procedures. For 
external monitoring, the team will contract a clinical research organization that will 
complete a pre-site trial assessment, quarterly monitoring visits during the trial 
implementation, and data collection closure visit. Additionally, Tampere University staff 
members will conduct bi-annual internal monitoring visits to the study sites. 
 
If requested, the investigators will make study documents (e.g., consent forms, drug 
distribution forms, case report forms) and pertinent hospital or clinic records readily 
available for inspection by the site auditors for confirmation of the study data if the study 
site is selected for auditing by the Comité D'Ethique de la FMPOS (Faculte de Medicine, 
de Pharmacie et D’Odonto-Stomatologie, Universite des Sciences, des Techniques et des 
Technologies de Bamako). 
 
 
4.30. Originally planned time schedule  
(not updated after version 1.0) 
 
In total, the project will take 4 years and 2 months. The detailed time schedule is below. 
 
November - December, 2019 
 
Finalising research plan 
Approval of the plan by appropriate institutional 

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review boards (IRB) 
 
Developing SOPs and planning practical 
arrangements  
 
 
 
 
 
Developing data entry and management system 
 
January – March 2020  
 
Finalising data entry and management system 
Staff recruitment and training 
Obtaining study drugs 
 
April 2020 – March 2021 
 
First study visits at all study villages 
 
April 2021 – December 2021 
 
Completion of 4 MDA rounds in all villages 
 
January 2022 
 
 
 
Interim analysis. Dropping placebo if justified 
 
January 2022 – September 2022  
Completion of all main study follow-up visits 
 
October 2022 – December 2022  
Data cleaning and analysis 
 
January 2023 – December 2023 
Write-up of main study results (mortality, 
morbidity, and growth outcomes 
Local results dissemination on main outcomes 
Completion of AMR follow-up visits and 
analyses, internationl and local results 
dissemination on AMR outcomes 
Continued feasibility analyses 
 
Roll-out of azithromycin MDA program, if 
indicated 
 
Figure 10 shows a graphic illustration of trial schedule. Planned interim analysis is 
marked in transparent red color. To shorten the trial period by six months, the last 
enrolled villages will be followed up only for 7 MDA rounds (30% of villages) or 6 
MDA rounds (20% of villages). 
 
 
 

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5. Statistical considerations and data analysis 
 
 
5.01. Specific hypotheses 
 
The main study hypotheses in terms of mortality effect are the following: 
1. Biannual azithromycin MDA to 1-11 month (29-364 days) old infants reduces 
their mortality 
2. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants reduces 
their mortality 
3. Quarterly azithromycin MDA has a bigger mortality effect than biannual MDA 
 
The study hypotheses in terms of other health effects are the following: 
4. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants reduces 
their 14-day period prevalence of fever with respiratory symptoms (ARI), fever 
without respiratory symptoms (malaria), and diarrhea 
a. Separately, there is a similar hypothesis on the effect of biannual azi MDA 
5. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants reduces 
their health facility visits for ARI, malaria, diarrhea, and other conditions  
a. Separately, there is a similar hypothesis on the effect of biannual azi MDA 
6. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants improves 
their length gain, and nutritional status (MUAC, WHZ)? 
a. Separately, there is a similar hypothesis on the effect of biannual azi MDA 
 
The study hypotheses in terms of intervention health effects on AMR are the following: 
7. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants does not 
cause a sustained increase in the prevalence of phenotypic azithromycin resistance 
among S. pneumoniae or E. coli strains isolated from their nasopharyngeal 
secretions or stools. 
a. Measured at 12 months after the intervention has stopped  
b. Separately, there is a similar hypothesis on the effect of biannual azi MDA 
8. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants does not 
increase the prevalence of azithromycin resistance among S. pneumoniae or E. coli 
strains isolated from 49–59-month-old children, who live in the same Malian 
communities but have not received azithromycin MDA? 
a. Measured when the intervention has lasted for 12-18 months 
b. Separately, there is a similar hypothesis on the effect of biannual azi MDA 
9. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants does not 
cause a sustained increase in the prevalence of azithromycin resistance genes in 
their intestinal microbiota. 
a. Measured when the intervention has lasted for 12-18 months  
b. Separately, there is a similar hypothesis on the effect of biannual azi MDA 
10. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants does not 
increase carriage of azithromycin resistance bacteria in treated infants 1 year after 

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their treatment has stopped, who live in a village where MDA intervention has also 
ceased. 
a. Measured after the intervention for individual child has stopped for 1 year  
b. Separately, there is a similar hypothesis on the effect of biannual azi MDA 
11. Quarterly azithromycin MDA to 1-11 month (29-364 days) old infants does not 
increase carriage of azithromycin resistance bacteria in treated infants 1year after 
their treatment has stopped, who live in a village where MDA intervention has 
continued. 
a. Measured after the intervention for individual child has stopped for 1 year  
b. Separately, there is a similar hypothesis on the effect of biannual azi 
MDA. 
 
 
The study hypotheses in terms of azithromycin mechanism of action are the following: 
12. Azithromycin MDA reduces malaria prevalence and parasite density, and 
improves mean blood hemoglobin concentration among 4–11-month-old infants, 
within 14 days of the MDA 
a. For these analyses, the two azithromycin can be combined 
13. Azithromycin MDA reduces general inflammation (CRP) among 4–11-month-old 
infants, within 14 days of the MDA 
a. For these analyses, the two azithromycin can be combined 
14. Azithromycin MDA reduces intestinal inflammation (fecal neopterin, 
myeloperoxidase, and alpha-1-antitrypsin concentrations) among 4–11-month-old 
infants, within 14 days of the MDA 
a. For these analyses, the two azithromycin can be combined 
 
There is no predefined hypothesis about the impact of biannual or quarterly azithromycin 
MDA to 1–11-month-old infants on mortality among 12–59-month-old children living in 
the same communities, but we will measure group-level differences on this as exploratory 
analysis. 
 
There are also no predefined hypotheses about the feasibility, acceptability, or equity of 
the azithromycin MDA intervention. 
 
 
5.02. Analytical approach 
 
The study team will express mortality in the three groups as number of deaths per 1000 
person-years at risk (deaths/PYR) and AMR as the proportion of E. coli or S. pneumoniae 
isolates that have reduced susceptibility (R or I category) towards azithromycin. For 
pairwise comparisons between any of the two groups, the team will calculate incidence 
rate ratio and its 95% confidence interval (CI) for mortality and risk ratio and its 95% CI 
for AMR prevalence at 12 and 24 months after the onset of the azithromycin MDA 
intervention.  
 
As an exploratory analysis, the team will also assess mortality (deaths / 1000 years at 
risk) among children who were 12–59-month-old at the time of the previous study drug 
administration in their village of residence. There is no predefined hypothesis for this 

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analysis, but it can provide information about possible herd protection generated through 
targeted azithromycin MDA.  
 
Mixed-effect Poisson regression model will be used to estimate the intervention effects 
on mortality, with random intercepts for the clusters. For twins, no adjustment for inter-
dependence will be made, i.e. we plan to analyse the data assuming independence 
between infants from the same mother. This is because our previous methodological 
research has demonstrated that analytic models that assume independence are robust in 
situations where cluster size is small and only a few of the analysable units are clustered 
(such as 1-2% twin rate, Xu et al., 2014). As per the closed-testing method for controlling 
multiplicity arising from multiple group comparisons, global null hypothesis of mortality 
in all three groups being the same will be tested at 5% significance level. Pairwise null 
hypothesis can be rejected only if the global null hypothesis has been rejected. Pairwise 
incidence rate ratio and corresponding 95% confidence intervals will be estimated. 
 
For the main analysis, we will analyse the mortality data of infants/children who receive 
azithromycin/placebo at the age of 1-11 months (29-364 days) at the beginning of each 3-
month period. Some of the person-time and deaths may occur at age 12-14 months during 
the 3-month periods. This is consistent with the study aim to assess the effect of MDA 
giving at the age of 1-11 months (29-364 days). As a secondary analysis, we will use the 
Lexis expansion approach to partition the follow-up time and deaths into intervals of 1-5, 
6-11, and 12-14 months of age (and also 1-11 and 12-14 months) and cross-classified 
with intervals defined according to the 3-month periods. This allows for more precise 
estimation of age-specific intervention effects and more precise adjustment for the time 
period effects. 
 
For AMR, the team will calculate the risk differences (and their two-sided 95% CIs) for 
AMR prevalence in azi-quarterly and azi-biannual groups as compared to the control 
group, separately. If the entire 95% CI of the risk difference of a MDA regime is below a 
predefined non-inferiority margin of 0.1 (meaning less than 10%-point absolute increase 
in AMR prevalence), the sample findings will be considered supportive of the hypothesis 
of no clinically significant increase in AMR prevalence for that regime. 
 
For morbidity analyses, we will calculate 14-day period prevalence of fever with 
respiratory symptoms (ARI), fever without respiratory symptoms (malaria), and diarrhea 
in the three intervention arms and do a three-group comparison using likelihood ratio 
tests. The latter will be obtained from mixed-effects logistic regression models, which 
accounts for the clustering of observations. If the test shows a statistically significant 
difference (p<0.05) between the three groups, we will proceed into three pair-wise 
comparisons, providing relative risks (RR) and their 95% Cis. Hypothesis testing in these 
pairwise comparisons will be done with likelihood ratio test obtained from mixed-effects 
logistic regression models. If the p-value is <0.05, the results will be considered 
supportive of the trial hypothesis. 
 
For growth analysis, we will calculate anthropometric indices LAZ (length for age Z-
score), WAZ (weight for age Z-score), WLZ (weight for length Z-score), and MUAC-Z 
(mid upper arm Z-score) for each participant as described in chapter 4.10. We will then 
calculate mean (SD) group values for each variable. In analyzing differences between 
groups, controlling for multiplicity will be conducted as per the closed-testing procedure, 

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by testing a global null hypothesis at 5% significance level. If the global null hypothesis 
is rejected (p<0.05), three pairwise comparisons will be carried out. The differences 
between the three groups and their 95% CIs will be obtained from mixed-effects 
regression models, that account for the clustering of observations. The global p-value will 
also be obtained from these models. 
 
For mechanism of action analyses, we will calculate the prevalence of malaria 
parasitemia as well as mean plasma CRP, blood hemoglobin and other biomarker 
concentration before and at two weeks after the MDA. We will then test a global 
hypothesis about differences between the groups using mixed-effects models, which 
account for clustering, for continuous variables and proportions, respectively with 
different, suitable link functions. If the global test shows a statistically significant 
difference (p<0.05) between the three groups, we will proceed into three pair-wise 
comparisons, providing the difference (95% CI) in means or proportions. If the p-value 
obtained from mixed-effects models is <0.05, the results will be considered supportive of 
the trial hypothesis. 
 
 
 
All children will be analysed in the group into which they were initially randomized, i.e. 
on an intention-to-treat basis. 
 
 
5.03. Frequency of analyses 
 
The randomisation code will be broken after the ninth study visit has been completed in 
all clusters (24 months after the last cluster was included in the study) and the database 
has been reviewed for data accuracy. The main analysis will be done at this point. The 
analysis will first be completed using letter-coded intervention names (A, B, C). Once the 
principal investigators have reviewed the analysis results and agreed that there is no need 
for further data or analysis edits, the code linking specific interventions to the letter codes 
will be opened.  
 
As described in chapter 4.13, one interim efficacy analysis will be completed when 60% 
of the planned post-MDA 3-month intervals have been completed. If there is a 
statistically significant mortality difference between both of the azithromycin groups and 
the placebo group but not between the two azithromycin groups, the placebo arm will be 
discontinued, and trial completed with two arms only. If there is also a statistically 
significant difference between the two azithromycin groups, the trial will be stopped, and 
the team will offer to work with Malian Ministry of Health to provide azithromycin MDA 
in the trial site and two other regions of Mali. 
 
Concomitantly with the interim efficacy analysis, there will be an interim safety analysis.  
 
Additional safety or efficacy analyses will be completed if requested by the study DSMB. 
 
 

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5.04. Sample size and its justification 
 
The sample size will be in total 1150 clusters (villages), of which we will use all for the 
primary analyses on mortality and secondary and tertiary samples for the secondary 
outcome analyses. At each round of home visits, each cluster is expected to contain on 
average 39 infants, of whom 34 will be found and given MDA and 31 found in the 
subsequent visit, thus providing data to the mortality analysis. Thus, approximately 
35,650 infants will provide follow-up data at first six home visit rounds, 28,520 on the 7th 
round and 17,820 on the 8th round. In total the trial will result in approximately 260,200 
analysable time intervals.  
 
The sample size is based on the following assumptions: 
1. Mortality among 1-11 month (29-364 days) old children in control group of 20 
deaths / 1000 PYR (20% lower than in the latest DHS) 
2. Mortality in the azi-biannual intervention group 16/1000 (20% relative reduction) 
3. Mortality in the azi-quarterly intervention group 12 / 1000 (25% lower than 
control, 40% lower than azi-biannual) 
4. 2-year intervention with 8 quarterly cycles of MDA for 50% of clusters, 7 rounds 
for 30%, 6 rounds for 20%), due to staggered entry but equal stopping time 
5. One interim analysis planned, when 60% of the planned 3-month time intervals 
have been completed 
6. One-sided 2.5% type 1 error, controlling of multiple group comparisons by the 
closed testing procedure and multiple looks by the Peto method.  
7. Coefficient of variation (k=sd/mean) of 0.1 in mortality among clusters. 
8. Unequal number of infants per cluster: on average 22 infants in a small village 
and 70 in a big village  
9. Unequal ratio of clusters. Control vs azi-biannual vs azi-quarterly = 3 : 4 : 2 
 
Using the power-by-simulation approach stratified by size of the clusters (dichotomized 
with cut-off point of 100 infants per cluster), it is determined that a sample size of 1150 
clusters with on average 31 analysable infants per cluster / time interval, there will be 
approximately 89% power for testing the hypothesis that biannual azithromycin MDA 
will reduce mortality, >99% power for testing the hypothesis that quarterly azithromycin 
MDA will reduce mortality and 80%  power for testing a hypothesis that quarterly 
azithromycin MDA will reduce mortality more than biannual azithromycin MDA.  
 
The relative risk reduction (RRR) of 20% in the azi-biannual group is modelled on the 
MORDOR-trial, in which the point-estimate for mortality reduction among under-one-
year old infants was 25% (in all the study countries) (Keenan et al, 2018). We estimate 
this reduction be slightly lower in Mali, because of the seasonal malaria chemoprevention 
that will be offered to 3-11-month-old infants in the Mali but was not practiced at the 
MORDOR sites. The 40% RRR for the azi-quarterly group is also modelled on 
MORDOR result indicating that in that sample practically all the mortality effect was 
concentrated in the first 3 months after the MDA (Porco et al, 2019). 
 
For the AMR data collection, the sample size will be 60 clusters or 1,350 children per 
time-point. This number will be tested from 4–14-month-old children at 12 months after 
the intervention has stopped and from 49–59-month-old children when 8 rounds of MDA 
have been completed. At the three other time points, we will test 450 children (taken 

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randomly from 20 clusters / arm). In total, this will mean 5,400 AMR-analyses for S. 
pneumoniae and the same number for E. coli. 
 
The AMR sub-sample size is based on the following assumptions 
1. 95% E. coli and 65% S. pneumoniae recovery rate from the collected samples 
2. AMR prevalence of 12% in the control group for E. coli and 6% for S. 
pneumoniae. These figures come from analysis of azithromycin resistance in 50 
E. coli and 50 S. pneumoniae samples from Mali in the so called ABCD trial.  
3. Non-inferiority margin of 10%-points in AMR prevalence 
4. 80% power, one-sided 2.5% type 1 error rate for each pairwise comparison 
against placebo control 
5. Coefficient of variation of 0.3 in AMR among clusters. 
 
For the analyses on azithromycin mechanism, we are using a convenience sample, i.e. all 
available children in the intended age-brackets (and avoiding repeated venous blood 
collection from the same children), in the study clusters where the AMR work is done. 
The sample size of 1,000 – 1,350 / analysis, or a mean of 333 - 450 / intervention arm, 
will provide these analyses 80% power at 5% two-sided type 1 error rate to detect an 
effect size of 0.25 – 0.30, assuming an intra-cluster correlation coefficient of 0.03 and a 
design effect of 1.6 to 2. 
 
For intra-household analyses there are no pre-set hypotheses, but the data will be used for 
descriptive purposes. 
 
 
5.05. Subgroup analyses 
 
For hypothesis generating purposes, we plan to carry out exploratory tests for interaction 
between the MDA intervention and the selected baseline variables, using the mortality 
and growth outcomes. Tests of hypotheses concerning intervention effects in these 
subgroups will be performed only if the interaction tests give statistically significant 
results (p<0.1). 
 
The following variables would be assessed potential effect modifiers 
1. Infant age at the time of MDA (1-5 months vs 6-11 months) 
2. Infant weight-for-age, length-for-age, weight-for-length at the time of MDA 
3. Infant sex 
4. Season of MDA dosing (malaria versus non malaria season) and SMC 
implementation in village (SMC given versus SMC not given) 
5. Cluster level coverage of SMC 
6. Cluster level baseline mortality (established at first census) 
7. Cluster and individual level coverage and number of administered azithromycin 
MDA doses 
8. District of residence 
9. Distance from the nearest health facility 
10. Household asset or income index 
11. Household WASH index 
12. National outreach strategy category (standard/advanced) 

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13. Hard-to-reach village status assessed by the field workers 
 
 
6. Ethical considerations 
 
 
6.01. General principles 
 
The trial will be performed according to Good Clinical Practice guidelines (ICH-GCP) 
and it will adhere to the principles of Helsinki declaration and regulatory guidelines in 
Mali. Trial recruitment shall not begin before the ethics committee in Mali (Comité 
D'Ethique de l’USTTB (Université des Sciences, des Techniques et des Technologies de 
Bamako) has given a favourable statement on it. An ethics committee in Finland has no 
legal mandate to authorize trials abroad, but a favourable opinion from one (Pirkanmaan 
Sairaanhoitopiirin eettinen toimikunta) will be required for TAU researchers to 
participate in the project. 
 
 
6.02. Informed consent 
 
Permissions for trial implementation will be sought on three levels at the study site: 
Village (cluster), household, and individual child.  
 
For village enrollment, members of the study team will visit village leadership, give them 
oral and written information about the trial (Appendix 1a) and request a permission to 
make an information campaign and perform study activities in the selected village. The 
response will be given verbally and documented electronically in a study database 
(Appendix 1b). 
 
If a village level permission is granted, the study team will run an information campaign 
about the upcoming trial in the village and its nearby health facilities. After one week of 
general information campaign, the team will start household visits. 
 
A team consisting of a community volunteer and a study team member will conduct the 
household visits. At each household visit, the team members will inform the heads of 
household and other household members about the purpose and processes of the trial, 
provide a written documentation about it (Appendix 2a) and request a consent (Appendix 
2b) to visit the household and collect trial data on a total of nine times over a period of 
two years. The response will be given verbally and documented electronically in a study 
database. If the person giving consent is illiterate, study personnel will ask her / him to 
invite an impartial witness to verify the consent. The name and home village of the 
impartial witness will be recorded in the consent database. 
 
Since the local language (Bambara) is mostly not a written language, many people may 
not be able to read it. For standardisation purposes, the team will write and hand out the 
participant information and consent form texts both in French and in Bambara. From the 

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Bambara text, the trial team will make an audiotape, so that the message can be given in a 
standardised manner by different data collectors. For information purposes, the team will 
also make a picture booklet as well as Bambara language videos describing the study 
procedures. 
 
During the household visits, the community volunteer and the data collector request a 
permission to weight and provide study drugs to all 1-11- month-old children (age 29-364 
days). Information about the purpose and process for this treatment is provided verbally 
and the response is also provided verbally and documented electronically in a study 
database (Appendix 3). 
 
At selected visits, sub-samples of children are invited for further data collection on 
secondary outcomes and other relevant data at a nearby health center or at central 
location in a village. Participant information and consent forms for the secondary 
outcome data collection are shown as Appendices 4a (participant information) and 4b 
(consent form). 
 
 
6.03. Possible risks and benefits to study participants 
 
Azithromycin stimulates smooth muscle contraction and may cause mild abdominal 
discomfort, vomiting, or diarrhea in some children. In earlier MDA trials with single-dose 
azithromycin use in infants, this has not been frequently reported by caregivers (Porco et 
al., 2009; Keenan et al., 2018; Chandramohan et al., 2019). In several studies, where 
azithromycin treatment has been compared to other antibiotics, the incidence of 
gastrointestinal side-effects has been smaller in the azithromycin than the comparison 
groups (Roord et al., 1996; Ferwerda et all., 2001; Langly at al., 2004). Hence, we don’t 
expect much of this problem for LAKANA participants.  
 
Among newborns, there is one theoretical safety concern, because of which neonates will 
be excluded from study drug administration in the LAKANA trial. In this age group, 
azithromycin may induce pyloric stenosis, an obstruction of gastric outlet that may 
require surgical intervention. In some population analyses, an increased risk for this rare 
condition has been associated with azithromycin use among under-14-day-old neonates, 
but not older children (Eberly et al., 2015). Since it is not known if the increased pyloric 
stenosis risk has been eliminated by one month of post-natal age also among preterm 
babies and since we will not be able to ascertain the duration of pregnancy at birth, all 
infants whose weight is below 3.0 kg will be excluded from the trial (3.0 kg is the 3rd 
weight centile among healthy 1 month olds, according to WHO growth charts).  
 
Besides the possibly increased risk of the rare pyloric stenosis in newborns and possibly 
causing abdominal discomfort, azithromycin has an excellent safety profile and is not 
expected to pose health risks to LAKANA participants. In the United States, 
azithromycin has been FDA approved for use only for children who are six-months or 
older, but there are numerous scientific reports and a systematic review that document 
safety also for younger infants (Pinto et al., 2012; McCallum et al., 2013; Beigelman et 
al., 2015; Smith et al., 2015). There are also several official US guidelines that 
recommend azithromycin use for under-six month old infants (American Academy of 

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Pediatrics, 2012; Centers for Disease Control, 2019). Also, in trachoma control programs, 
azithromycin has been used for post-neonatal infants for 20 years without safety 
concerns.  
 
All participants will benefit from trial participation in terms of repeated home visits, 
during which caregivers may ask health-related questions from the study personnel. The 
study staff making home visits will not be medically trained and they will be instructed 
not to provide direct medical advice. However, if needed, they will facilitate referral to a 
local health facility. If a further referral to a hospital is required, the study team will assist 
in transfer where necessary. For children participating in the mechanistic substudy for 
whom blood samples are collected and Hb and CRP levels measured on site: on the first 
visit, if a child is asymptomatic or has mild respiratory symptoms, and the CRP is above 
or Hb is below a critical threshold based on reference ranges from the Mali national and 
local guidelines in use at that time, the child will be given the study drug and then 
actively referred to the nearest health clinic. Transportation will be ensured. On the 
second visit two weeks later, if the Hb reaches a critical threshold based on reference 
ranges from the Mali national and local guidelines in use at that time, the child will be 
actively referred to the nearest health clinic and transportation will be ensured. Upon 
referral, a paper copy of an instruction leaflet to health clinics will be given to the 
caregiver to show at the clinic (Appendix 5). 
 
Two thirds of the participants will receive azithromycin MDA, which is expected to 
reduce mortality and offer other health benefits to the participants. At the end of the trial, 
if the analysis suggests a mortality reduction by azithromycin, all participants who are 1-
11 months (29-364 days) old, will receive 20 mg / kg azithromycin. 
 
 
6.04. Amount of collected blood and other biospecimens 
 
From most participants, there will be no biological specimen collection.  
 
At four time points, for a sub-sample of approximately 1,350 4-14 month old children 
and the same number of 49-59 month old children, we will also collect rectal and 
nasopharyngeal or rectal swabs. At each time point, the children will be different, so for 
each child, there will only be one sampling. Additionally, at visit 11 we will sample 15-
26-months old children who have received 1-4 doses of MDA 1 year earlier in villages 
that have stopped MDA. In the Koulikoro or Kati (tertiary AMR sample) region we will 
sample 24-35 months old children who have received 1-4 doses of MDA 1 year earlier 
and who live in a village that has continued MDA. This will allow us to assess whether 
azithromycin resistant bacteria survive in individuals that have previously been treated. 
We will use flocculated swabs for the nasopharyngeal and stool samples. The estimated 
mass of the collected stool is 200 ug.  
 
From a subgroup of 6–8-month-old or 12-14 month old infants and young children, there 
will be one venous blood collection, with a total collected blood volume of maximum of 
5 ml. The collected volume represents approximately 1% of the infant’s total blood 
volume and it is replaceable from the infants’ own blood production within 24 hours. 
From the same individuals, we will collect 2-10 g of stool and 5-10 ml of urine 

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6.05. Insurance coverage 
 
The Malian trial team will purchase an insurance that will cover professional liabilities 
for the study team and all medical costs for study participants, caused by their 
participation in LAKANA trial. 
 
Any adverse reactions arising during the study will be reported to the study team through 
the appropriated process and then referred into the national health system. Medical costs 
will be covered by the study budget. 
 
The team will be provided with security services when deemed necessary by national 
security advice. Tampere University will purchase additional security services for all 
expatriate staff, consisting of travel advice, security briefings and assistance in medical 
and other emergencies. All visiting study team members and guests will be required to 
abide by security advice and procedures.   
 
 
6.06. Compensation to study participants 
 
For most LAKANA participants, the scheduled home visits (9 in total) will take 
approximately 20 –30 minutes. For time used on these visits, the households will not be 
compensated. 
 
For children participating in sub-samples, participants will be invited to a nearby health 
facility or central location in a village and visits will include collection of detailed data on 
household economic, biological specimen collection or measuring anthropometrics. Such 
visits are expected to take approximately an hour. For these visits, the participants will be 
compensated with approx. $2-$5 or another amount deemed appropriate by the local 
study coordination team to cover transport. If blood is drawn, some food and drink will 
be offered.  
 
For children participating in sub-samples and screened for Malaria at visit 9, the study 
nurses will provide the result of the rapid diagnostic test to the caregivers. The nurses will 
actively refer the children with a positive test result to the nearest health clinic where they 
can receive the appropriate care according to the Mali national treatment guidelines in use 
at that time. The health clinics will be compensated by the study team on a regular basis 
for the treatment costs incurred. 
 
6.07. Payments to the trial organisers  
 
Trial organisers do not receive any incentives for enrolling participants to the study. 
During the trial period, they will receive their normal salary from their employers or a 
personal stipend supporting postgraduate studies and living expenses. 
 

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6.08. Data safety and monitoring board (DSMB) 
 
A review board will be constituted prior to commencement of the study to oversee the 
progress and assess the safety of the intervention. The trial team will provide the DSMB 
members a monthly summary of trial progress and documented and expected numbers of 
serious adverse events (SAEs).  
 
The DSMB will meet in person before the trial onset and conduct thereafter scheduled 
electronic meetings 1-2 times a year, to discuss the progress and safety data. 
Additionally, any member of the DSMB may request an additional meeting at any point. 
 
As part of the adaptive design, there will a planned interim data analysis, as explained in 
chapter 4.13. In case of any unusual events, members of the DSMB may request 
additional interim analyses at their discretion. 
 
 
6.09. Stopping rules for the study  
 
The DSMB will convene before the onset of the trial to agree on specific stopping rules 
for the trial.    
Tentatively, DSMB may recommend termination, dropping the placebo arm, or other 
modification of the study if: 
1. In an interim analysis, there is strong evidence of a mortality benefit (reduced 
mortality) from the study intervention. The DSMB will agree on the exact 
definition of “strong evidence”, but according to the Peto-rule, in an interim 
analysis a mortality difference should be considered statistically significant only if 
the 2-sided p-value is lower than approximately 0.001 (Peto et al., 1976). 
Simulation studies have shown that the Peto’s rule is not overly extreme and 
results in practically no inflation of type 1 error in the final analysis (Freidlin et 
al., 1999). 
2. In an interim analysis, there is strong suspicion of harm (increased mortality or 
incidence of other SAEs) from the study intervention. For harm assessment, no 
fixed statistical rules will be applied, but the DSMB will holistically consider 
point estimates and confidence intervals for mortality / SAE incidence 
differences, p-values from appropriate statistical tests and other relevant factors, 
when determining its recommendation about study continuation or 
discontinuation.  
3. In an interim analysis, there is strong evidence of futility (mathematical 
expectation predicting from the accrued data that the major study hypotheses 
cannot be supported with statistical significance with the originally planned 
sample size). 
 
While stopping rules will be utilized to help the DSMB to assess the justification of trial 
continuation, the DSMB will consider the balance of risks and benefits as well as 
consistency with external evidence. Thus, the DSMB recommendation to continue or 

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discontinue will not be based on any single issue or value in any of the analyses but a 
comprehensive analysis considering multiple issues around the trial. 
 
 
 
7. Personnel, resources, and study management 
 
 
7.01. Research institutions 
 
The main participating research institutions are CVD-Mali in Mali (CVD-Mali), Tampere 
University in Finland (TAU), University College London in UK (UCL) and Tro Da Ltd, a 
UK-based global health consulting company.  
 
The project will be implemented by CVD-Mali (Mali), Tampere University (Finland), 
University College London (UK), and Tro Da Ltd (UK). The partners will all contribute 
to trial design, implementation, analysis, and reporting. For their specific contributions, 
TAU will act as the coordinating institution and main grantee and provide expertise in 
clinical trials, social science, and certain laboratory analyses, CVD-Mali will bear the 
main responsibility of the trial implementation and provide knowledge on research and 
health in Mali, UCL will coordinate the AMR and economics work and provide expertise 
on these subjects and azithromycin mechanisms, and Tro Da Ltd will provide expertise in 
MDA platforms and support the team in trial implementation and internal 
communication.  
 
All the participating organizations have extensive experience from conducting large 
clinical trials in Africa, coordinating international networks of researchers, and managing 
large multi-site grants. The principal investigators (PIs) and the Co-PIs have also earlier 
experience from working together: the Mali and TAU PIs on another clinical trial 
involving azithromycin treatment in Mali and the UCL and TAU ones from multiple 
projects in Malawi and elsewhere in East Africa. 
 
All the organizations are equipped with necessary research infra-structure in their main 
offices. For trial implementation, CVD-Mali will need to review health facilities in the 
Kayes region and likely refurbish some office and basic laboratory facilities (including 
e.g. freezer purchase). TAU and UCL will need to identify 1-2 new scientists, but 
otherwise their project personnel have already been identified. At CVD-Mali, there is 
also a need to identify 1-2 more scientists to work on the project. In addition, there is a 
need to hire the entire team of data collectors and field supervisors at CVD-Mali. 
 
 
7.02. Principal investigators 
 
Dr. Per Ashorn (overall PI) has coordinated the design of the LAKANA trial and will 
bear the overall responsibility of its completion. He will also act as a supervisor to 
Finnish or Malian students involved in the project and enrolling for post-graduate studies 

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at Tampere University. Dr. Per Ashorn serves as a Professor of Paediatrics and Director 
of the Center for Child Health Research at Tampere University. He is a medical specialist 
in paediatrics and paediatric infectious diseases, and he has carried out research on child 
health and nutrition in low-income settings for more than 25 years. Dr. Per Ashorn has 
formal training in and extensive experience from designing, coordinating and reporting 
clinical trials, including two on the use of azithromycin to promote child health.  
 
Dr. Samba O. Sow (CVD-Mali co-PI) will be responsible for the practical 
implementation of the trial in Mali. He serves as Director-General of the Center for 
Vaccine Development (CVD) in Mali and Professor of Medicine at the School of 
Medicine, University of Maryland (USA), where he has coordinated clinical trials and 
epidemiological studies on a range of vaccine-preventable diseases. A medical doctor and 
epidemiologist, he has received numerous honors, including the French Legion of Honor 
and National Order of Merit and Officer in 2017 for his work on the Ebola epidemic. He 
was the Commemorative Fund Lecturer of the American Society of Tropical Medicine 
and Hygiene in 2006 and held the post of Minister of Health for Mali from April 2017 
until May 2019.  
 
Dr. Nigel Klein (UCL co-PI) will lead the MDA antimicrobial resistance component in 
the LAKANA trial. He works as a Professor and Consultant in Pediatric Infectious 
Diseases and Immunology at Great Ormond Street Children’s Hospital (GOH), London, 
and Professor of Infection and Immunity at the UCL Great Ormond Street Institute of 
Child Health (ICH). He has established and led the Infectious Diseases and Microbiology 
Unit at ICH the Department of Infection at UCL and was recently appointed as Lead of 
the Paediatric Programme at the Wellcome Trust funded African Health Research 
Institute in KwaZulu-Natal. Dr. Klein has been working in the field of Infectious 
Diseases for more than 30 years, published more than 400 research papers and supervised 
over 60 PhD students. He has a particular interest in pathogenesis of infection and 
conducts also microbiome research, including two studies with azithromycin and other 
antibiotic interventions during pregnancy in low-income settings. He is key to a newly 
established Precision AMR Centre at UCL, GOH and ICH that provides infrastructure for 
both molecular and phenotypic technologies to examine antimicrobial resistance and will 
support capacity building in Mali where needed. 
 
Dr. Ulla Ashorn (TAU co-PI) will coordinate the sub-study on MDA equity, feasibility 
and acceptability, contribute to all other parts of the trial and bear the responsibility of 
international program management. She will also act as a supervisor to Finnish or Malian 
students involved in the project and enrolling for post-graduate studies at Tampere 
University. Dr. Ulla Ashorn is a social scientist with background in public and global 
health. She currently works as a Senior Scientist at the Center for Child Health Research 
at Tampere University. She has conducted research on women’s and children’s health 
mainly in East Africa. She is familiar with both qualitative and quantitative health 
research methods as well as policy and politics research including stakeholder analysis 
and engagement.  
 
Dr Camilla Ducker (Tro Da co-PI) will be working with CVD-Mali on implementation, 
communication, and helping to build the capacity of personnel recruited and assisting on 
the equity, feasibility and acceptability sub-study. Dr. Ducker is a clinical doctor and 
public health consultant. She launched her own global health company Tro Da Ltd in 

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2018, which has successfully carried out work for the Bill and Melinda Gates Foundation 
(REACH program), the Task Force for Global Health (laboratory capacity strengthening), 
and the World Health Organization. Prior to that, Camilla worked for the UK 
Government’s Department of International Development, managing the majority of its 
Neglected Tropical Disease programs. Camilla Chairs the Neglected Tropical Disease 
Equity Working Group.  
 
Dr. Yin Bun Cheung will act as the principal statistician on the project and advise other 
biostatisticians at Tampere University on analytic strategy, statistical programming and 
interpretation of the trial findings and provide troubleshooting where necessary. Dr. 
Cheung is a biostatistician with background in paediatric epidemiology and infectious 
disease clinical trials. He serves as a Professor at the Centre for Quantitative Medicine at 
Duke-NUS Medical School, Singapore. He is also an Adjunct Professor of International 
Health at the Tampere University, Finland. He has been the principal investigator of 
multiple research projects funded by the National Medical Research Council, Singapore, 
to improve statistical methods in infectious disease research. He has studied child health 
in Asian and African countries and is the author of Statistical Analysis of Human Growth 
and Development (2014) and co-author of Survival Analysis: A Practical Approach 
(2006).  
 
7.03. Study statisticians 
 
Dr. Yin Bun Cheung will act as the principal statistician on the project. Data review and 
cleaning, as well as data and metadata annotation and public posting will be completed by 
two biostatisticians at Tampere University, Ms. Lotta Hallamaa and Mr. Juho Luoma. 
They both have an MSc degree in biostatistics and experience from data cleaning and 
analysis. Ms. Hallamaa is currently completing her PhD on long-term follow-up of 
Malawian children, whose mothers were treated with azithromycin in pregnancy. 
 
 
7.04. Other main scientists and their responsibilities 
 
Dr Elaine Cloutman-Green will ensure high standard laboratory practice and diagnostic 
stewardship for the AMR studies. She is the National Institute of Health Research 
national diagnostic infection lead in the UK. She sits on the UK AMR diagnostic 
stewardship committee and is a Principal Clinical Scientist at Great Ormond Street 
Hospital (GOSH), leads the Precision AMR facility at GOSH and holds a clinical 
lectureship at UCL. 
 
 
7.05. Other employed staff 
 
There will be a study team based in Bamako for overall supervision of the trial. This team 
will be made up of a trial project manager, data managers, administrators, and financial 
managers. 
 

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In additional to the central Bamako office, there will be a regional office in Kita City and 
6-9 district offices. These will employ a supervisor who will supervise a group of data 
collectors.  
 
At each of the study districts, there will be several field teams that will implement the 
azithromycin MDA and collect study data. Each team will be made up of one GCP-
trained data collector and at least two relais workers (community volunteers) who will act 
as community drug distributors in conjunction with a data collector. In total, the team will 
hire one hundred and eighty data collectors for the main study and each of them will 
cover 3-6 villages (together with the community volunteers). 
 
For the analyses on morbidity, growth, AMR, azithromycin mechanisms, feasibility, cost 
and safety, an additional research team will be posted in the four health facilities where 
data collection on these additional variables takes place. The team will consist of a nurse, 
two anthropometrists and one motorcycle messenger. The nurse will collect 
biospecimens, monitor health facility visits by trial participants, and interview families 
about possible side-effects. The anthropometrists will measure child size, interview 
families about feasibility and economics, and help the nurse in other issues. The 
motorcycle messenger will help gather the participants and deliver biospecimens daily to 
the study laboratories in Kita.  
 
Biological samples collected within LAKANA are planned to be primarily shipped to a 
site-laboratory that the LAKANA team will establish in the city of Kita. In this 
laboratory, two laboratory assistants will do primary processing and storage of the 
samples. For further analyses, the samples will be shipped to CVD-Mali laboratory in 
Bamako. For high-quality analyses, all laboratories will be provided with high quality 
laboratory equipment. Laboratory personnel specific to the project will be recruited 
locally and capacity built by the national and international institutions involved in the 
trial.  
 
Most of the employed study personnel will be Malians as many as possible data 
collectors and supervisors will be hired locally. This will ensure the trust of the 
population who are receiving the intervention and contribute to local capacity building. 
 
 
7.06. Arrangements for day-to-day management of the study 
 
The overall, general management of the study will be coordinated by the lead PI and 
Tampere University. Operational and day-to-day management will be the responsibility 
of CVD-Mali in collaboration with Tro Da.  
 
The study team at CVD-Mali will have a management group, led by the Malian co-PI and 
responsible for trial implementation. The management group will meet weekly, to review 
progress reports on enrollment and follow-up, to discuss any problems encountered and 
to accept a general plan of activities for the subsequent week. A data manager will 
produce weekly progress reports of the success of enrollment and follow-up as well as 
data quality, for use by the core group. 
 

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A CVD-Mali project manager will produce weekly duty lists for data collectors and 
supervisors, using custom-made computer software that is linked to the trial database. 
S/he and her sub-ordinates will distribute these plans to members of the study team at the 
end of each week. Once a month, the study team will have a larger staff meeting to 
discuss the progress and any acute issues related to the study. 
 
 
7.07. Communication plan 
 
For external communication, there will be a trial website in national and international 
languages to cascade information. The study team will also prepare an electronic 
quarterly newsletter, to be shared with local partners, Ministry of Health and the wider 
scientific community. Tampere University will prepare regular progress reports to the 
funder, as agreed in the grant contracts.  
 
In addition to the website and quarterly newsletters, the team will organize a national 
stakeholder meeting once a year and regional stakeholder meetings as needed. Before 
onset of data collection, there will be multiple launches of the trial at district level. 
During the study, there will be district level and village level community meetings to 
distribute information. There will also be a system to allow village elders and CSCOM 
staff to raise issues through the CDDs and Data collectors, so that local additional level 
meetings can be organised as needed, to deal with rumours or any other community 
anxieties.   
 
The internal trial communication will be coordinated and conducted by the study team in 
Mali. Data Collectors will meet with their supervisors on a weekly basis and feedback 
any issues and the central team in Bamako will receive weekly reports from each office 
and feedback also. The report will include highlights, activities, risks and future activities 
planned.   
 
Leaflets in local languages and using infographics will be developed and tested in the 
community to communicate the trial to the communities. 
 
 
7.08. Project Steering Group  
 
LAKANA trial implementation will be overseen by a 5-member project steering group 
(PSG) that will consist of the overall PI and the four co-PIs from the organizations that 
will receive the project funding (Ashorn, Sow, Klein, Ashorn, Ducker). The PSG will 
meet electronically at 1-2-week intervals at the beginning of data collection and later at 1-
2-month intervals, to review progress and plan for future. A trial statistician from TAU 
will produce monthly progress reports of the success of enrollment and follow-up as well 
as data quality, for use by the PSG. 
 
The PSG will make all the key strategic decisions over the trial, i.e. it will decide 
submission of the project plan and reports the funder, to IRBs, and to other key 
authorities. The PSG will also make decisions on amendments to trial protocol, project 

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personnel or budget, or data ownership and use. 
 
 
7.09. National and international advisory groups 
 
There will be two advisory groups to the study team: A National Advisory Committee 
(NAC) and an international Technical Advisory Group (TAG).  
 
The NAC will consist of approximately 10 health professionals, who will advise the 
Research team on general design issues, implementation strategy, conduct and progress 
of the trial. It will also be consulted to provide advice on any ad-hoc national issues that 
arise during the trial. The NAC will provide insights to the team especially regarding 
stakeholder interests and priorities as well as other relevant initiatives taking place at the 
study site. The NAC will also provide technical advice, assist with resolving issues and 
risks especially at a community level, use influence and authority to assist the project in 
achieving its outcomes, and communicate about the project in their respective 
organizations. 
 
Approximately once a year, the NAC will organize a wider stakeholder meeting. This 
meeting, attended by 100 – 150 participants especially from the study area, will serve 
mostly an information purpose, but also, to help the study team with details of trial 
planning and implementation and related communication in the communities. 
 
The TAG will consist of three members, who are internationally recognized experts in the 
fields of child survival, clinical trials, mass drug administration, or antimicrobial 
resistance. It will advise the PIs and co-PIs on general design issues, implementation 
strategy, conduct and progress of the trial. It will also be consulted to provide advice on 
any ad-hoc technical issues that arise during the trial. It will discuss DSMB reports and 
recommendations and advise the trial team on their implementation. 
 
 
 
8. Training provided 
 
The study will offer numerous training opportunities to Malian and other undergraduate, 
graduate and post-graduate students, but no specific projects have yet been identified. 
The budget includes a 4-year remuneration for one Malian PhD student who would 
tentatively study the feasibility and acceptability questions. 
 
 
 
9. Possible constraints 
 
Heavy rains between June and September may reduce human mobility and access to 
some locations and hence complicate the MDA and data collection. The team will, 
however, make extra preparations with rain gear etc and hence the delays or missed visits 

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are expected to be non-significant.  
 
Safety (for the population and the research team) is another possible constraint. The 
Northern part of Mali is politically instable and there is a safety concern in that area. 
Most of Kayes Region, where the study is being planned is, however, stable and there is 
no reason to expect a change in that situation. CVD-Mali has budgeted for security 
support for internal travel. 
 
 
 
10. Funding 
 
Tampere University has received funding for the proposed trial from the Bill & Melinda 
Gates Foundation (Seattle, WA, USA). For implementation, Tampere University will 
issue subawards to CVD-Mali, UCL, and Tro Da Ltd. 
 
 
 

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Infectious Diseases Project: Measles Vaccine Initiative  Enteric Disease Project   
Pneumococcus, Meningococcus, & Haemophilus influenzae Prevention Program 
Malaria Project: Bandiagara Malaria Project    Malaria Training Program 
12. Appendices 
 
Appendix 1a: Information about the trial to village leadership  
(English version, to be translated into French and Bambara and read aloud to village leaders 
 
 
 
 
 
CVD-MALI  
Large-scale Assessment of the Key health-promoting Activities of New 
mass drug administration regimens with Azithromycin (LAKANA) 
 
Information for Participants and Informed Consent Forms for village 
leaders 
This document contains information for villages participating in a cluster-randomised, 
double-blinded, parallel group, controlled trial, to test the effects of mass drug 
administration of azithromycin on mortality and other outcomes among 1-11-month-old 
infants in rural Mali. It also contains the necessary consent forms for participants to enrol 
in the trial. 
 
Key Investigators 
 
Centre pour le Développement des Vaccins–Bamako, Mali 
Per Ashorn, MD, PhD 1, Ulla Ashorn, PhD 1, Yin Bun Cheung, PhD 1,2, Camilla Ducker, 
MSc., MBBS 3, Nigel Klein, MBBS, PhD 4, Samba O. Sow MD., MSc., FASTMH 5. 
 
Participating Academic Institutions 
 1 Tampere University, Finland; 2 Duke-NUS Medical School, Singapore; 3 Tro Da 
Ltd, UK; 4 University College London, UK; 5 CVD-Mali, Bamako, Mali. 
CENTRE POUR LE DEVELOPPEMENT DES VACCINS, MALI 
 
Director General: Samba Ousmane SOW, M.D., M.Sc., FASTMH 
 
Professor of Medicine 
 

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Study Overview 
Your village is invited to participate in the LAKANA trial, in which we will try to 
determine if a drug called azithromycin can help Malian infants to enjoy better health and 
better rates of survival. In the study, some infants will receive azithromycin and others 
will receive something called a placebo, which looks and tastes like azithromycin but 
does not contain any medicine. The study will compare these different groups of children. 
Azithromycin is an effective and safe medicine that is commonly used to treat illnesses – 
it has been used for many years to successfully and safely treat infections, including 
millions of cases of an eye infection called trachoma. There are studies that show that 
periodically using this medicine to treat healthy children in West Africa might reduce 
mortality. The LAKANA trial team is investigating if such treatment would also be 
beneficial to Malian infants.  
In total, we plan to include 50–60 000 children in the study, from approximately 1150 
villages or urban areas in the Kayes, Kita and Koulikoro regions. If you give consent for 
your village to participate, the LAKANA team will visit the village every three months 
over the next two years, to ask you some questions, and give study medicine to all 1-11 
month old infants living in the village. 
Occasionally, children who take azithromycin may suffer stomach upsets for a short 
period, but otherwise we don’t expect the treatment to cause any harmful side effects. 
Reactions, although rare, are always possible when medicines are given to children. So 
we will monitor the health of all the children who receive the medicine. In the unlikely 
case that a child in your village experiences a negative reaction, CVD-Mali will help that 
child to receive medical care and cover any costs related to that care.  
This study could lead to major improvements in the health of infants in Mali. By 
participating in this trial, your village may be contributing to the development of better 
health programmes for the whole country. If this trial is successful, it is possible that 
azithromycin may be given to infants across Mali and West Africa.  
You do not have to give permission for your village to take part in this study – you are 
free to decide if the village should participate or not.  
What is the study about? 
Azithromycin is a type of medicine, an antibiotic, that is commonly used to treat 
infections, including eye infections that cause blindness. Recent research studies have 
shown that infants in certain African countries had better survival rates when they took 
azithromycin every six months, when compared to those who did not take the medicine. 
In the LAKANA trial, researchers from the Centre pour le Développement des Vaccins-
Mali (CVD-Mali) and their partners in Finland and the United Kingdom are studying how 
well azithromycin works in Mali. The trial is being funded by an American organization 
called the Bill & Melinda Gates Foundation. 
The LAKANA trial will be carried out in 1150 villages and urban areas in the Kayes, Kita, 

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and Koulikoro regions, if local representatives have given permission for study activities 
to take place there. After receiving permission, in each village or town area, the study team 
will visit all households every three months for a period of two years. The team members 
will ask for permission to record and store a list of household members. If there are any 1-
11 month old infants in the household, the household will be offered the chance to 
participate in the study and the infants living there will be offered the study medicine. At 
subsequent visits, team members will monitor the effects of the study medicine by asking 
questions about the infants’ health. 
Two types of study mixture will be given to infants in the LAKANA trial: azithromycin 
and placebo. Placebo is something that looks and tastes like azithromycin but does not 
contain any active ingredient that can treat infection. In one third of the study villages, all 
infants will receive azithromycin during all the study visits, every three-months. In 
another third of the study villages, infants will receive azithromycin at half of the visits 
and placebo at the other half. In the last third of the study villages, infants will always 
receive placebo. 
Deciding which villages would be in which group was done by chance, like taking one 
single grain from a bag full of rice. During the study nobody will know whether the 
infants in your village are receiving placebo or active azithromycin at any of the home 
visits – this information will only be available later on to the study team. This way of 
carrying out the trial will help the study team to make correct conclusions about the 
health effects of azithromycin. 
Study outcomes 
The study will test to see if azithromycin has a positive impact on infant health and if it 
reduces infant mortality. The results of the study will be used to influence public health 
policy in Mali and in the wider sub-Saharan African region.  
At approximately midway during the trial, researchers will analyze the obtained results. If 
there are clear indications that azithromycin is having a positive effect on mortality and 
the general health of infants, the trial will be modified, and azithromycin will then be 
given to all infants in the selected areas – and in other regions of Mali.  
Who can take part in this study? 
All households in the villages selected for the study will be offered the chance to join the 
study, for the purposes of collecting data. At each of the quarterly visits to households 
taking part in the study, all 1-11 month-old (age 29-364 days) infants will be offered the 
study medicine, as long as the infant has no known intolerance to azithromycin.  
What will happen in the study? 
If your village takes part in this study, the study team will visit the village four times a 
year for two years (eight times in total, after an initial visit to collect information). At 
each visit, we will ask participants questions about their households and their infants’ 
health and illnesses, we will measure the weight of 1-11 month old infants, and will give 
them an appropriate dose (one teaspoonful or less) of study medicine. The medicine will 

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come in the form of a mixture and will be placed in the infant’s mouth using a disposable 
syringe.  
Since the study visits will take place at three-month intervals and only 1-11 month old 
(age 29-364 days) infants will be treated, the maximum number of treatments given to 
any individual infant will be four. 
The first visit will take approximately 45 minutes, the subsequent visits will likely take 
approximately 15 minutes each. 
If any household or child enrolled in the study is not at home when the study team comes 
to visit, they will try to visit again later or they may call to agree on a time when the missing 
people will be at home. If for some reason the visit cannot be completed, infants can still 
continue to take part in the study and will be seen during subsequent home visits. 
How do I give consent for trial participation? 
As representatives of your village, you will be asked for permission to carry out trial-
related activities in the village. If we receive your permission, we will start the study 
activities. First of all, at each household, we will ask for permission to visit the household 
and ask some questions every three months for the next two years. Additionally, on each 
visit, we will ask for parents’ or guardians’ permission to weigh 1-11 month old infants 
and administer study medicine to them.  
You don’t need to sign any documents to give consent for the trial to take place in your 
village but we will record your consent on our computer. We will also ask an independent 
person to witness your consent if you are not literate. His or her name will also be 
recorded in the computer system. 
Is participation voluntary? 
Your village’s participation in this study is entirely voluntary – for the village as a whole 
and for each individual household/child within the village. Your village does not have to 
take part in this study. Once enrolled, individual participants in the study will be free to 
leave the study at any time without giving a reason. Infants’ participation will not change 
anything about the health care they receive at any health facilities. If, for any reason, 
infants are no longer available for follow-up visits, we will ask to contact them again to 
check on their health. Participants may refuse this as well. If a participant withdraws from 
this study, we will keep the information that we have already collected from infants in 
your village. 
We will tell you about any significant new findings that are available during the study 
that may affect your willingness to involve infants in your village in the study. 
Can villages / infants be removed from this study? 
The study doctors and Malian, Finnish, or UK-based research committees overseeing the 
safety and rights of study participants, can remove your village and/or any infants in the 
village from the study or even stop the entire study without your approval if serious 

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health problems are noticed. If a parent or guardian decides to stop an infant’s 
participation in the study, or if an infant’s participation is ended, the study doctor or study 
staff may still ask the parents/guardian about that infant’s participation in the study. 
Answering any such questions is, however, voluntary. 
What are my village’s responsibilities if we take part in this study? 
If you agree to your village’s participation in this research, we will visit households every 
three months and ask villagers to inform us if they move or expect to be unavailable. If 
villages and individuals agree to take part Taking part in this study does not change 
responsibilities with regard to other non-study related health care for infants in the 
village. 
What risks are involved in azithromycin use and trial participation? 
Azithromycin is commonly used and is known to be safe for infants. Even so, a few 
people may react to azithromycin and experience brief episodes of diarrhoea, nausea, 
abdominal pain and vomiting. A very small number of children may also experience liver 
problems, rashes, itching, or swelling of the lips or throat when they take azithromycin.  
In extremely rare cases, some young infants may experience a problem where they have 
trouble passing food from the stomach to the intestines, which can cause vomiting and 
may require surgery. This can happen to any infant, whether they take azithromycin or 
not. If an infant experiences a reaction like this or has developed other symptoms, care 
should be sought urgently at the closest health facility and the study doctor should also be 
contacted.  
If an infant in your village falls ill because of the study treatment, CVD-Mali will pay for 
the costs of medical treatment according to Malian standards of care.  
What are the possible benefits of being in this study? 
There may be no direct benefits to the people in your village as a result of participating in 
this study. However, the information obtained in this study will be important in deciding 
if azithromycin could improve the health of infants in Mali and other similar countries. 
What kind of feedback can we expect from research results? 
The results of the study will be known after it is finished. The study team will hold 
meetings to inform the community about the results. 
What are the costs of taking part in this study? 
All research activities will be performed free of charge. 
What are the payments for being in this study? 
There are no payments for taking part in the study. But infants in your village will be 
visited every three months while the study is active, and participants may signal other 
issues related to infants’ health to the relais and data collector who will visit you. 

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What are the alternatives to participating in this study? 
Giving azithromycin to infants in Mali is not currently part of routine care. The 
alternative to participating in the study is choosing not to take part. 
How will information be kept private? 
To protect the privacy of the parents/guardians and children living in your village, we 
will keep the information collected by the study secure. We will only allow authorized 
people to see it, including the study team from Mali, UK, and Finland, their 
representatives and the Ethics Committee in Mali, who act to protect people who take 
part in research. If study results are released to the public, the identities of participants 
will not be shared.  
Who can answer questions about this study? 
If you have any questions regarding your infant’s participation or if an infant has a 
problem as a result of participating in this study, you may contact at any time Dr 
Fatoumata Diallo on (00223) 74 60 18 19,  Dr Fadima Cheick Haidara on (0023) 66 73 
34 91, Prof Samba Sow on (00223) 76348947, or CVD-Mali, CNAM, Ex-Institut 
Marchoux on (00223) 20 23 60 31.  
To learn more about the ethical approval of this study or about your rights as a research 
subject, you can contact the Ethics Committee of USTTB at (00223) 2022 5277, Prof. 
Saïbou Maïga at (00223) 66750118  or Prof. Mahamadou Diakité (Permanent Secretary) 
at (00223) 66231191 
 
 

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Protocol version 7.0: 13. November. 2024 
 
Appendix 1b: Verbal Consent form for Village Participation 
To be recorded electronically by a member of the LAKANA study team, on behalf of the 
head of the village chiefs, representatives or other persons authorised to speak on behalf 
of a village/grouping of hamlets 
The giving and recording of consent should be confirmed by an independent witness 
 
The persons named below affirm that they have given verbal consent and received 
information about the proposed trial or had this information explained to them, and 
that they consent to all eligible infants living in their village being enrolled in the 
trial. Witnesses to the consent process affirm that consent was given and 
information about the trial received according to the conditions laid out in the 
information about the trial. 
 
 
Name of village 
 
_______________________                _                                                                                   
Name of Person authorised to give consent on behalf of village  
  
_______________________________                          _                                                       
Position / Role 
 
 
Did the above-named person give consent?      
Yes/No  
Date 
 
 
 
 
Name of Witness to Consent Procedures: _______________                                                
(If subject is illiterate, or otherwise unable to give consent) 
 
 
______________________________________________________________________ 
Witness’s Confirmation 
Yes/No 
Date 
 
 
Name of Investigator: ________________________                                                             
or Authorized Representative obtaining informed consent 
 
 
________________________________________________________________________
 
 
Investigator’s Confirmation (initials or study code) 
Yes/No 
 
Date 
 

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Protocol version 7.0: 13. November. 2024 
 
Infectious Diseases Project: Measles Vaccine Initiative  Enteric Disease Project   
Pneumococcus, Meningococcus, & Haemophilus influenzae Prevention Program 
Malaria Project: Bandiagara Malaria Project    Malaria Training Program 
Appendix 2a: Information about the trial to a potential participant  
(English version, to be translated into French and Bambara and read aloud to village leaders 
 
 
 
 
CVD-MALI  
Large-scale Assessment of the Key health-promoting Activities of New 
mass drug administration regimens with Azithromycin (LAKANA) 
 
Information for Participants and Informed Consent Forms for 
households 
This document contains information for participants in a cluster-randomised, double-
blinded, parallel group, controlled trial, to test the effects of mass drug administration of 
azithromycin on mortality and other outcomes among 1-11-month-old infants in rural 
Mali. It also contains the necessary consent forms for participants to enrol in the trial. 
 
Key Investigators 
 
Centre pour le Développement des Vaccins–Bamako, Mali 
Per Ashorn, MD, PhD 1, Ulla Ashorn, PhD 1, Yin Bun Cheung, PhD 1,2, Camilla Ducker, 
MSc., MBBS 3, Nigel Klein, MBBS, PhD 4, Samba O. Sow MD., MSc., FASTMH 5. 
 
Participating Academic Institutions 
 1 Tampere University, Finland; 2 Duke-NUS Medical School, Singapore; 3 Tro Da 
Ltd, UK; 4 University College London, UK; 5 CVD-Mali, Bamako, Mali. 
CENTRE POUR LE DEVELOPPEMENT DES VACCINS, MALI 
 
Director General: Samba Ousmane SOW, M.D., M.Sc., FASTMH 
 
Professor of Medicine 

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Protocol version 7.0: 13. November. 2024 
 
Study Overview 
You are invited to participate in the LAKANA trial, in which we will try to determine if a 
drug called azithromycin can help Malian infants to enjoy better health and better rates of 
survival. In the study, some infants will receive azithromycin and others will receive 
something called a placebo, which looks and tastes like azithromycin but does not contain 
any medicine. The study will compare these different groups of children. 
Azithromycin is an effective and safe medicine that is commonly used to treat illnesses – 
it has been used for many years to successfully and safely treat infections, including 
millions of cases of an eye infection called trachoma. There are studies that show that 
periodically using this medicine to treat healthy children in West Africa might reduce 
mortality. The LAKANA trial team is investigating if such treatment would also be 
beneficial to Malian infants.  
In total, we plan to include 50–60 000 children in the study, from approximately 1150 
villages or urban areas in the Kayes, Kita, and Koulikoro regions. If you agree to 
participate, the LAKANA team will visit you every three months over the next two years, 
to ask you some questions, and give study medicine to 1-11 month old infants living in 
your household. 
Occasionally, children who take azithromycin may suffer stomach upsets for a short 
period, but otherwise we don’t expect the treatment to cause any harmful side effects. 
Reactions, although rare, are always possible when medicines are given to children. So 
we will monitor the health of your child(ren) after they receive the medicine. In the 
unlikely case that your child experiences a negative reaction, CVD-Mali will help the 
child to receive medical care and cover any costs related to that care.  
This study could lead to major improvements in the health of infants in Mali. By 
participating in this trial, you may be contributing to the development of better health 
programmes for the whole country. If this trial is successful, it is possible that 
azithromycin may be given to infants across Mali and West Africa.  
You do not have to take part in this study – you are free to decide if you want to 
participate or not.  
What is the study about? 
Azithromycin is a type of medicine, an antibiotic, that is commonly used to treat 
infections, including eye infections that cause blindness. Recent research studies have 
shown that infants in certain African countries had better survival rates when they took 
azithromycin every six months, when compared to those who did not take the medicine. 
In the LAKANA trial, researchers from Centre pour le Développement des Vaccins-Mali 
(CVD-Mali) and their partners in Finland and the United Kingdom are studying how well 
azithromycin works in Mali. The trial is being funded by an American organization called 
the Bill & Melinda Gates Foundation. 
The LAKANA trial will be carried out in 1150 villages or urban areas in the Kayes, Kita, 

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Protocol version 7.0: 13. November. 2024 
 
and Koulikoro regions, after local representatives have given permission for study activities 
to take place there. In each village or town area, the study team will visit all households 
every three months for a period of two years. The team members will ask for permission 
to record and store a list of household members. If there are any 1-11 month old infants in 
the household, the household will be offered the chance to participate in the study and the 
infants living there will be offered the study medicine. At subsequent visits, team members 
will monitor the effects of the study medicine by asking questions about the infants’ health. 
Two types of study mixture will be given to infants in the LAKANA trial: azithromycin 
and placebo. Placebo is something that looks and tastes like azithromycin but does not 
contain any active ingredient that can treat infection. In approximately one third of the 
study villages, all infants will receive azithromycin during all the study visits, every 
three-months. In another third of the study villages, infants will receive azithromycin at 
half of the visits and placebo at the other half. In the last third of the study villages, 
infants will always receive placebo.  
Deciding which villages would be in which group was done by chance, like taking one 
single grain from a bag full of rice. During the study neither you nor anyone in the study 
team will know whether your infant receives placebo or active azithromycin at any of the 
home visits – this information will only be available later on to the study team. This way 
of carrying out the trial will help the study team to make correct conclusions about the 
health effects of azithromycin. 
Study outcomes 
The study will test to see if azithromycin has a positive impact on infant health and if it 
reduces infant mortality. The results of the study will be used to influence public health 
policy in Mali and in the wider sub-Saharan African region.  
At approximately midway during the trial, researchers will analyze the obtained results. If 
there are clear indications that azithromycin is having a positive effect on mortality and 
the general health of infants, the trial will be modified, and azithromycin will then be 
given to all infants in the selected areas – and in other regions of Mali.  
Who can take part in this study? 
All households in the study area will be offered the chance to join the study for the 
purposes of collecting data. At each of the quarterly visits to households taking part in the 
study, all 1-11 month old infants will be offered the study medicine, as long as the infant 
has no known intolerance to azithromycin.  
What will happen in the study? 
If you take part in this study, the study team will visit you four times a year for two years 
(eight times in total, after an initial visit to collect information). At each visit, we will ask 
you some questions about your household and your infants’ health and illnesses, we will 
measure the weight of your 1-11 month old infants, and will give them an appropriate 
dose (one teaspoonful or less) of study medicine. The medicine will come in the form of a 
mixture and will be placed in the infant’s mouth using a disposable syringe.  

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Protocol version 7.0: 13. November. 2024 
 
Since the study visits will take place at three-month intervals and only 1-11 month old 
infants will be treated, the maximum number of treatments given to any individual infant 
will be four. 
The first visit will take approximately 45 minutes of your time, the subsequent visits will 
likely take approximately 15 minutes each. 
If you are not at home when the study team comes to see you, they will try to visit again 
later or they may call you to agree on a time when you will be at home. If for some reason 
the visit cannot be completed, you and your infant can still continue to take part in the study 
and be seen during subsequent home visits. 
How do I give my consent for trial participation? 
Before starting any trial-related activities in your home village, we will have received 
permission to conduct trial activities there from your village representatives. At your 
household, first of all, we will ask for permission to visit you and ask some questions 
every three months for the next two years. Additionally, on each visit, we will ask your 
permission to weigh your 1-11 month old infants and administer study medicine to them.  
You don’t need to sign any documents for your participation, but we will record your 
consent on our computer. We will also ask an independent person to witness your consent 
if you are not literate. His or her name will also be recorded in the computer system. 
Is participation voluntary? 
Your infant’s participation in this study is entirely voluntary. You do not have to take part 
in this study, and you are free to leave the study at any time without giving a reason. 
Your infant’s participation will not change anything about the health care your infant 
receives at any health facilities. If, for any reason, you are no longer available for follow-
up visits, we will ask to contact you again to check on the health of your infant. You may 
refuse this as well. If you withdraw from this study, we will keep the information that we 
have already collected from your infant. 
We will tell you about any significant new findings that are available during the study 
that may affect your willingness to involve your infant in the study. 
Can my infant be removed from this study? 
The study doctors and Malian, Finnish, or UK-based research committees overseeing the 
safety and rights of study participants, can remove you or your infant from the study or 
even stop the entire study without your approval if serious health problems are noticed. If 
you decide to stop your infant’s participation in the study, or if your infant’s participation 
is ended, the study doctor or study staff may ask you some questions about your infant’s 
participation in the study. Answering any such questions is, however, voluntary. 
What are my responsibilities if I take part in this study? 
If you participate in this research, we will visit your household every three months and 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
ask you to inform us if you move or expect to be unavailable. If you give consent for your 
infant to participate in this study, you will still be responsible for all other non-study 
related health care for your infant. 
What risks are involved in azithromycin use and trial participation? 
Azithromycin is commonly used and is known to be safe for infants. Even so, a few 
people may react to azithromycin and experience brief episodes of diarrhoea, nausea, 
abdominal pain and vomiting. A very small number of children may also experience liver 
problems, rashes, itching, or swelling of the lips or throat when they take azithromycin.  
In extremely rare cases, some young infants may experience a problem where they have 
trouble passing food from the stomach to the intestines, which can cause vomiting and 
may require surgery. This can happen to any infant, whether they take azithromycin or 
not. If you think your infant is experiencing a reaction like this or has developed other 
symptoms, please seek care urgently at the closest health facility and contact the study 
doctor.  
If your infant falls ill because of the study treatment, CVD-Mali will pay for the costs of 
medical treatment according to Malian standards of care.  
What are the possible benefits of being in this study? 
There may be no direct benefits to you or your infant as a result of participating in this 
study. However, the information obtained in this study will be important in deciding if 
azithromycin could improve the health of infants in Mali and other similar countries. 
What kind of feedback can I expect from research results? 
We will know the results of the study after it is finished. The study team will hold 
meetings to inform the community about the results. 
What are the costs of taking part in this study? 
All research activities will be performed free of charge. 
What are the payments for being in this study? 
There are no payments for taking part in the study. But you and your infant will be visited 
every three months while the study is active, and you may signal other issues related to 
your infant’s health to the relais and data collector who will visit you. 
What are the alternatives to participating in this study? 
Giving azithromycin to infants in Mali is not currently part of routine care. The 
alternative to participating in the study is choosing not to take part. 
How will information be kept private? 
To protect your privacy, we will keep the information collected by the study about your 
infant secure. We will only allow authorized people to see it, including the study team 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
from Mali, UK, and Finland, their representatives and the Ethics Committee in Mali, who 
act to protect people who take part in research. If study results are released to the public, 
the identities of participants will not be shared.  
Who can answer questions about this study? 
If you have any questions regarding your infant’s participation or if your infant has a 
problem as a result of participating in this study, you may contact at any time Dr 
Fatoumata Diallo on (00223) 74 60 18 19,  Dr Fadima Cheick Haidara on (0023) 66 73 
34 91, Prof Samba Sow on (00223) 76348947, or CVD-Mali, CNAM, Ex-Institut 
Marchoux on (00223) 20 23 60 31.  
To learn more about the ethical approval of this study or your rights as a research subject, 
you can contact the Ethics Committee of USTTB at (00223) 2022 5277, Prof. Saïbou 
Maïga at (00223) 66750118 or Prof. Mahamadou Diakité (Permanent Secretary) at 
(00223) 66231191. 
 

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Protocol version 7.0: 13. November. 2024 
 
Appendix 2b: Verbal Household Consent form for Trial Participation 
(English version, to be translated into French and Bambara and read aloud to village leaders 
 
To be recorded electronically by a member of the LAKANA study team, on behalf of the 
head of the household or other person authorised to give consent for household 
participation 
The giving and recording of consent should be confirmed by an independent witness 
 
The persons named below affirm that they have given verbal consent and received 
information about the proposed trial or had this information explained to them, and 
that they consent to all eligible infants living in the household being enrolled in the 
trial. Witnesses to the consent process affirm that consent was given and 
information about the trial received according to the conditions laid out in the 
information about the trial. 
 
 
 
Name of household 
 
_______________________________                          _                                                       
Name of Person giving consent for household  
  
_______________________________                          _                                                      
Role in household  
 
 
Did the above-named person give consent?      
Yes/No  
Date 
 
 
 
Name of Witness to Consent Procedures: _______________                                         
(If subject is illiterate, or otherwise unable to give consent) 
 
______________________________________________________________________ 
Witness’s Confirmation 
Yes/No 
Date 
 
 
Name of Investigator: ________________________                                                             
or Authorized Representative obtaining informed consent 
 
_____________________________________________________________________
 
 
Investigator’s Confirmation (initials or study code) 
Yes/No 
 
Date 

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Protocol version 7.0: 13. November. 2024 
 
Appendix 3: Consent to provide study medicine to an individual infant 
(English version, to be translated into French and Bambara and read aloud to village leaders 
 
For Parent/Guardian aged 18 years or older or married Parent/Guardian aged 16 
years or older 
[If parent or guardian is younger than 18 and unmarried, or younger than 16 years of age, 
skip to next page] 
 
The persons named below affirm that they have given verbal consent and received 
information about the proposed trial or had this information explained to them, and 
that they consent to the infant or infants in their care being enrolled in the trial, to 
receive study medicine and for information about these infants to be collected by the 
trial team. Witnesses to the consent process affirm that consent was given and 
information about the trial received according to the conditions laid out in the 
information about the trial. 
 
_______________________________                          _                                                       
Name of Household & identifier for Infant receiving study medicine 
  
_______________________________                          _                                                       
Name and Role of Person giving consent 
 
 
 
Did the above-named person give consent? 
Yes/No  
 
Date 
 
Name of Witness to Consent Procedures: _______________                                                
(If subject is illiterate, or otherwise unable to give consent) 
 
____________________________________________________________________ 
Witness’s Confirmation 
 
Yes/No 
 
 
Date 
 
Name of Investigator: ________________________                                                             
or Authorized Representative obtaining informed consent 
 
______________________________________________________________________
 
 
Investigator’s Confirmation (initials or study code) 
 
Yes/No 
 
 
Date 
 
 
 

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Protocol version 7.0: 13. November. 2024 
 
Appendix 3: Consent to provide study medicine to an individual infant 
(English version, to be translated into French and Bambara and read aloud to village leaders 
 
Informed Consent Form for Parents/Guardian of participants younger than 16 years 
old, or unmarried participants younger than 18 years old 
 
The persons named below affirm that they have given verbal consent and received 
information about the proposed trial or had this information explained to them, and 
that they consent to the infant or infants in their care being enrolled in the trial, to 
receive study medicine and for information about these infants to be collected by the 
trial team. Witnesses to the consent process affirm that consent was given and 
information about the trial received according to the conditions laid out in the 
information about the trial. 
 
_______________________________                          _                                                       
Name of Household & identifier for Infant receiving study medicine 
  
_______________________________                          _                                                       
Name and Role of Person giving consent 
 
 
 
Did the above-named person give consent? 
Yes/No  
 
Date 
 
Name of Witness to Consent Procedures: _______________                                                
(If subject is illiterate, or otherwise unable to give consent) 
 
____________________________________________________________________ 
Witness’s Confirmation 
 
Yes/No 
 
 
Date 
 
Name of Investigator: ________________________                                                             
or Authorized Representative obtaining informed consent 
 
______________________________________________________________________
 
 
Investigator’s Confirmation (initials or study code) 
 
Yes/No 
 
 
Date 
 
 
 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
Infectious Diseases Project: Measles Vaccine Initiative  Enteric Disease Project   
Pneumococcus, Meningococcus, & Haemophilus influenzae Prevention Program 
Malaria Project: Bandiagara Malaria Project    Malaria Training Program 
Appendix 4a: Information on secondary outcome data collection 
(English version, to be translated into French and Bambara and read aloud to village leaders 
 
 
 
 
 
 
CVD-MALI  
Large-scale Assessment of the Key health-promoting Activities of New 
mass drug administration regimens with Azithromycin (LAKANA) 
 
Information about Participation in the Anti-Microbial Resistance and 
other sub-studies and Informed Consent Forms for individuals  
This document contains information for participants in a cluster-randomised, double-
blinded, parallel group, controlled trial, to test the effects of mass drug administration of 
azithromycin on mortality and other outcomes among 1-11-month-old infants in rural 
Mali. It also contains the necessary consent forms for participants to enrol in the trial. 
 
Key Investigators 
 
Centre pour le Développement des Vaccins–Bamako, Mali 
Per Ashorn, MD, PhD 1, Ulla Ashorn, PhD 1, Yin Bun Cheung, PhD 1,2, Camilla Ducker, 
MSc., MBBS 3, Nigel Klein, MBBS, PhD 4, Samba O. Sow MD., MSc., FASTMH 5. 
 
Participating Academic Institutions 
 1 Tampere University, Finland; 2 Duke-NUS Medical School, Singapore; 3 Tro Da 
Ltd, UK; 4 University College London, UK; 5 CVD-Mali, Bamako, Mali. 
CENTRE POUR LE DEVELOPPEMENT DES VACCINS, MALI 
 
Director General: Samba Ousmane SOW, M.D., M.Sc., FASTMH 
 
Professor of Medicine 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
Introduction 
Thank you for participation in the LAKANA trial, which will try to determine if a drug 
called azithromycin can help Malian infants to enjoy better health and better rates of 
survival.  
You will have already received information about the trial and have given consent for the 
trial team to ask questions about the health of the child/children in your care and for us to 
give them trial medicine (azithromycin or placebo). 
If you would like more information about the trial or be reminded of its key aims, please 
speak to the members of the trial team or the relais communautaire who visit your village 
or the study team.  
 
LAKANA sub-studies 
 
The main LAKANA study will be implemented in approximately 1150 villages or town 
areas in Kayes. In some of the villages, there will be additional sub-studies, in which we 
will collect further information on the effects and feasibility of azithromycin distribution. 
Your village and others around the CSComs of Kati, Bendougouba, Dafela, Djidian, 
Kofeba and Koulikoro have been selected for the sub-studies. Therefore, we would like to 
describe them to you now and ask if you are in principle willing to participate or allow 
your child to participate in some of them.  
In total, there will be four sub-studies that will be completed over the next 2-3 years: one 
on infant growth, second on the possible development of antimicrobial resistance, third 
on the mechanisms how azithromycin works and fourth on the feasibility of distributing 
azithromycin to populations in Mali. No child will be invited to participate in these sub-
studies more than once or twice. Later, when we are about to do any of these sub-studies, 
we will ask your permission again. 
 
Infant growth sub-study 
In some earlier studies, antibiotic treatment has improved child growth. Therefore, we 
would also like to study if infants who receive azithromycin are bigger than infants who 
receive placebo.  
To measure growth, we will at certain home visits invite all 6-8 month-old and 12-14 
month old children to a nearby health centre or central location in village. At the health 
centre/central location, we will measure the child’s weight, length, and arm. Growth 
measurement is harmless, although some infants cry a bit when their length is taken. This 
visit will take approximately 30 minutes plus your travel time to the location. 
 
The Antimicrobial resistance (AMR) sub-study 
When antibiotics are used, bacteria sometimes become resistant to them, leading to 
reduced options for treatment of children or adults with infections. In order to judge if 
this happens with azithromycin in Kayes, the trial team will conduct an antimicrobial 

Trial protocol: LAKANA 
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Protocol version 7.0: 13. November. 2024 
 
resistance (AMR) sub-study in your village. 
To measure AMR, the study team will at selected home visits collect swab samples from 
children who are 4-14 month-old or 49-59 month-old. The samples will be collected with 
a thin cotton swab from the nose and rectum of the child, at your home or a central 
location of your village. The procedure is harmless and takes only some minutes, 
although swabbing the nose may sometimes feel a bit uncomfortable and some children 
may cry a bit. Any irritation is, however, mild and transient. 
The AMR samples will be collected on four of the home visits that the LAKANA team 
will make: when the household joins the study and at 12, 24, and 36 months thereafter. 
Since the samples are taken only from children who are at the right age-bracket at these 
visits, the same child can provide samples only once and we will ask for your permission 
each time before taking any samples. 
The collected samples will be stored and analysed at CVD-Mali laboratory in Bamako. 
Selected samples will be shipped for further analyses in collaborating centres in UK, 
Finland and possibly elsewhere. All samples will be coded and stored anonymously.  
 
How azithromycin works 
In order to better understand how azithromycin works in infants, the study team will carry 
out two sets of data collections.  
At the fourth study visit, before provision of the study medicine, the study team will take 
a small blood sample with a finger-prick or heel-prick from all 4-11 month-old infants, at 
a central location of your village. The team will also collect a stool sample from the same 
infant. Two weeks later, you will be asked to bring the infant to a nearby health facility, 
for collecting another finger-prick or heel-prick blood sample and a stool sample. At this 
second visits, there will also be an interview about the health of the infant after she 
received the study medicine. 
Later, at selected home visits, we will invite 6-8 month-old and 12-14 month-old infants 
to be brought to a health centre or another central location in the village. At the health 
centre or  central location, a study nurse will collect a small blood sample (less than a tea-
spoon) from the child’s arm. Before the visit, you will be given a disposable diaper and a 
small plastic container. We will ask you to place the disposable diaper on the child on the 
morning of the health centre visit and the study team will collect a urine sample from it. 
In the container, we will ask you to collect a sample of the child’s stool and bring it to the 
health centre. There will also be an interview about the health of the infant after she 
received the study medicine. 
The health facility/central location visits will take approximately 45 minutes plus your 
travel time. The procedures during these visits are harmless, but blood sampling can 
cause some discomfort and the child may have mild tenderness and bruising following 
the blood being drawn. Very rarely children can feel lightheaded for a short period of 
time. 
Since the samples are taken only from children who are at the right age-bracket at 
selected visits, the same child can provide samples only once or at maximum twice and 
we will ask for your permission each time before taking any samples. 
The collected samples will be stored and analysed at CVD-Mali laboratory in Bamako. 

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Protocol version 7.0: 13. November. 2024 
 
Selected samples will be shipped for further analyses in collaborating centres in UK, 
Finland and possibly elsewhere. All samples will be coded and stored anonymously.  
 
Acceptability and feasibility of azithromycin distribution 
The study will also try to answer questions about the acceptability and feasibility of 
treating infant with azithromycin. In order to do so, the study team will ask questions 
about these themes during selected home visits. Questions will be about your infant’s 
health, about the local health system and about the acceptability of azithromycin to your 
community, for example. Your answers will help to shape the outcomes of the study.  
These additional questions will be asked at your home, by specifically trained study 
personnel. In total, there will be three interviews and each of them will take 
approximately 30 minutes. Your answers will be entirely confidential and will not be 
used to identify you or your family in any way. 
 
Is participation voluntary? 
Your infant’s participation in these sub-studies is entirely voluntary. You do not have to 
take part in these, and you are free to leave them at any time without giving a reason.  
 
What are the possible benefits of being in these sub-studies? 
There may be no direct benefits to you or your infant as a result of participating in these 
sub-studies. However, the information obtained in these sub-studies will be important in 
deciding if azithromycin could improve the health of infants in Mali and other similar 
countries. 
 
What kind of feedback can I expect from research results? 
We will know the results of the sub-studies when the trial has finished. The study team 
will hold meetings to inform the community about the results. 
 
What are the alternatives to participating in these sub-studies? 
These sub-studies are an addition to the main azithromycin trial and are not a routine part 
of the trial’s drug treatment. They are not either a part of routine health care. The 
alternative to participating in these sub-studies is choosing not to take part. Not 
participating in this sub study does not affect your right to participate in the main 
Azithromycin trial.  

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Protocol version 7.0: 13. November. 2024 
 
 
How will information and biological samples be kept private? 
To protect your privacy, we will keep the information collected by the sub-studies about 
your infant secure. We will only allow authorized people to see it, including the study 
team from Mali, UK, and Finland, their representatives and the Ethics Committee in 
Mali, who act to protect people who take part in research. If study results are released to 
the public, the identities of participants will not be shared.  
Biological samples taken from your child will also be stored securely at the CVD 
laboratory in Mali. All samples will be coded and stored anonymously.  
 
Who can answer questions about these sub-studies? 
If you have any questions regarding your infant’s participation or if your infant has a 
problem as a result of participating in this study, you may contact at any time Dr 
Fatoumata Diallo on (00223) 74 60 18 19,  Dr Fadima Cheick Haidara on (0023) 66 73 
34 91, Prof Samba Sow on (00223) 76348947, or CVD-Mali, CNAM, Ex-Institut 
Marchoux on (00223) 20 23 60 31.  
To learn more about the ethical approval of this study or your rights as a research subject, 
you can contact the Ethics Committee of USTTB at (00223) 2022 5277, Prof. Saïbou 
Maïga at (00223) 66750118  or Prof. Mahamadou Diakité (Permanent Secretary) at 
(00223) 66231191. 
 

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Appendix 4b: Consent form for Participation in LAKANA Sub-studies 
(English version, to be translated into French and Bambara and read aloud to village leaders 
 
For Parent/Guardian aged 18 years or older or married Parent/Guardian aged 16 
years or older 
[If parent or guardian is younger than 18 and unmarried, or younger than 16 years of age, 
skip to next page] 
The persons named below affirm that they have given written consent and received 
information about the proposed LAKANA sub-studies or had this information 
explained to them, and that they consent to all eligible infants in their care being 
enrolled in the sub-studies. This includes consenting to the trial team taking all 
necessary biological samples. Witnesses to the consent process affirm that consent 
was given and information about the sub-studies received according to the 
conditions laid out in the trial information. 
I am consenting to:  
1. Participation in the growth sub study   
2. Participation in the antimicrobial resistance sub study    
3. Participation in the mechanisms sub study    
4. Participation in the acceptability and feasibility sub study    
 
_______________________________                          _                                                      
Printed name of participant  
  
_______________________________                          _                                                      
Printed name of participant’s parent/guardian 
 
    _______________ 
Signature or Fingerprint of Participant’s Parent/Guardian  
Date 
 
Name of Witness to Consent Procedures: _______________                                                
 

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(If subject is illiterate, or unable to sign) 
 
 
_____________________________________________________________________ 
Witness’s Signature  
 
Date 
 
 
 
Name of Investigator: ________________________                                                          
or Authorized Representative obtaining informed consent 
 
 
_____________________________                   _______________________________ 
Investigator’s Confirmation (initials or study code) 
Yes/No 
 
Date 
 
 

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Protocol version 7.0: 13. November. 2024 
 
Appendix 4b contd: Consent form for Participation in LAKANA Sub-studies 
(English version, to be translated into French and Bambara and read aloud to village leaders 
 
Informed Consent Form for Parents/Guardian of participants younger than 16 years 
old, or unmarried participants younger than 18 years old 
 
The persons named below affirm that they have given written consent and received 
information about the proposed LAKANA sub-studies or had this information 
explained to them, and that they consent to all eligible infants in their care being 
enrolled in the sub-studies. This includes consenting to the trial team taking all 
necessary biological samples. Witnesses to the consent process affirm that consent 
was given and information about the sub-studies received according to the 
conditions laid out in the trial information. 
I am consenting to:  
1. Participation in the growth sub study   
2. Participation in the antimicrobial resistance sub study    
3. Participation in the mechanisms sub study    
4. Participation in the acceptability and feasibility sub study    
 
_______________________________                          _                                                       
Printed name of participant  
  
_______________________________                          _                                                       
Printed name of participant’s parent/guardian 
 
    ______________ 
Signature or Fingerprint of Participant’s Parent/Guardian  
Date 
 
Name of Witness to Consent Procedures: _______________                                                 
(If subject is illiterate, or unable to sign) 
 
 

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Protocol version 7.0: 13. November. 2024 
 
 
__________________________________________________________________ 
Witness’s Signature  
 
Date 
 
 
Name of Investigator: ________________________                                                           
or Authorized Representative obtaining informed consent 
 
 
_____________________________________________________________________
 
 
Investigator’s Confirmation (initials or study code) 
Yes/No 
 
Date 
 
 

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Protocol version 7.0: 13. November. 2024 
 
Appendix 5, referral letter for asymptomatic children with abnormal 
laboratory values observed during a village visit 
(English version, to be translated into French)