Antibiotics for Children With Severe Diarrhoea
Phase: PHASE3
Conditions: Diarrhea
Sponsor: World Health Organization

ABCD trial protocol, version 9.0, 21 December, 2018 
Page 1 of 57 
 
 
Study protocol: Antibiotics for Children with Severe Diarrhoea (ABCD) Trial 
Overall Study Coordination 
World Health Organization, Maternal Newborn, Child and Adolescent Health Research 
and Development Team, Geneva, Switzerland 
Field Sites 
Centre pour le Développement des Vaccins (CVD-Mali), Bamako, Mali 
Kenya Medical Research Institute (KEMRI) 
Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania 
Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi 
International centre for diarrhoeal disease research, Bangladesh (icddr,b), Dhaka, 
Bangladesh 
Centre for Public Health Kinetics, Delhi, India 
Aga Khan University (AKU), Karachi, Pakistan 
WHO Coordinators: Dr. Jonathon Simon, Dr. Rajiv Bahl 
Principal Investigators: 
 
Bangladesh 
Dr Tahmeed Ahmed (icddr,b, Dhaka, Bangladesh) 
Dr Md. Jobayer Chisti (icddr,b, Dhaka, Bangladesh) 
India 
Dr Sunil Sawazal (Centre for Public Health Kinetics, Delhi, India) 
Dr. Usha Dhingra (Centre for Public Health Kinetics, Delhi, India) 
Kenya 
Dr. Benson Singa (Kenya Medical Research Institute, Nairobi, KE) 
Dr Judd L. Walson (University of Washington, Seattle, WA, USA) 
Malawi 
Dr Queen Dube (Queen Elizabeth Central Hospital, Blantyre, Malawi) 
Dr Naor Bar-Zeev (Johns Hopkins University, Baltimore, MD, USA and University of 
Liverpool, Liverpool, UK and Malawi-Liverpool-Wellcome Trust Clinical Research 
Programme, Blantyre, Malawi) 
Mali 
Dr Samba O Sow (CVD-Mali, CNAM, Bamako, Mali) 
Dr Karen L Kotloff (University of Maryland, Baltimore, MD, USA) 
Pakistan 
Dr. Farah Qamar (AKU, Karachi, Pakistan) 
Dr. Tahir Yousafzai (AKU, Karachi, Pakistan) 
Tanzania 
Dr Karim Manji (Muhimbili University of Health and Allied Sciences, Dar es Salaam, 
TZ) 
Dr Chris Duggan (Boston Children’s Hospital, Boston, MA, USA) 
 
 

ABCD trial protocol, version 9.0, 21 December, 2018 
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TABLE OF CONTENTS 
PROTOCOL VERSIONS AND AMENDMENT HISTORY .............................................. 5 
0. 
BACKGROUND ............................................................................................................... 6 
1. 
OBJECTIVES ................................................................................................................... 9 
1.1 
Co-Primary Objectives: ............................................................................................... 9 
1.2 
Secondary Objectives: ............................................................................................... 10 
2. 
SIGNIFICANCE AND IMPLICATIONS .................................................................... 10 
2.1 
Study sites .................................................................................................................. 11 
2.2 
Study Design .............................................................................................................. 11 
2.3 
Eligibility criteria ....................................................................................................... 11 
2.4 
Intervention ................................................................................................................ 12 
2.5 
Duration of follow-up ................................................................................................ 12 
3. 
RATIONALE FOR THE DEFINITION OF STUDY POPULATION AND 
ELIGIBILITY CRITERIA ................................................................................................... 13 
3.1 
Study Population ........................................................................................................ 13 
3.2 
Inclusion criteria ........................................................................................................ 13 
3.3 
Exclusion criteria ....................................................................................................... 13 
4. 
RECRUITMENT ............................................................................................................ 14 
4.1 
Screening ................................................................................................................... 14 
4.2 
Stabilization period .................................................................................................... 14 
4.3 
Consent ...................................................................................................................... 14 
4.4 
Enrolment .................................................................................................................. 14 
5. 
STANDARD MANAGEMENT AND INTERVENTION ........................................... 15 
5.1 
Study Drugs Intervention ........................................................................................... 15 
5.2 
Randomization and blinding ...................................................................................... 15 
5.3 
Study drugs administration ........................................................................................ 16 
6. 
FOLLOW UP .................................................................................................................. 17 
6.1 
Day 45 Follow up ...................................................................................................... 17 
6.2 
Day 90 Follow up ...................................................................................................... 17 
6.3 
Day 180 Follow up .................................................................................................... 18 
6.4 
Outcome measurements ............................................................................................. 18 
6.5 
Collection and processing of biological samples  ..................................................... 18 
7. 
SERIOUS ADVERSE EVENTS .................................................................................... 20 

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8. 
TRAINING ...................................................................................................................... 21 
9. 
TIMELINE ...................................................................................................................... 21 
10. DATA ANALYSIS ........................................................................................................... 21 
10.1 Primary Objectives .................................................................................................... 22 
10.2 Secondary Objectives ................................................................................................ 23 
10.3 Sample size ................................................................................................................ 25 
11. RISK MITIGATION ...................................................................................................... 27 
12 STUDY MANAGEMENT AND OVERSIGHT ........................................................... 29 
12.1 Coordination .............................................................................................................. 29 
12.2 Local Governance ...................................................................................................... 29 
12.3 Ethical Review ........................................................................................................... 30 
12.4 Trial Advisory Group and Technical Steering Committee ........................................ 30 
12.5 Data Safety and Monitoring Board ............................................................................ 30 
12.6 Quality assurance ....................................................................................................... 30 
12.7 Data management ...................................................................................................... 31 
12.8 References ................................................................................................................. 32 
13 SITE IMPLEMENTATION PLANS ............................................................................ 33 
13.1 Bangladesh ................................................................................................................. 33 
13.2 India ........................................................................................................................... 37 
13.3 Kenya ......................................................................................................................... 40 
13.4 Malawi ....................................................................................................................... 44 
13.5 Mali ............................................................................................................................ 47 
13.6 Pakistan ...................................................................................................................... 50 
13.7 Tanzania ..................................................................................................................... 53 
 

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STUDY OVERVIEW 
Although the current WHO recommended management package for acute diarrhoea (ORS, zinc 
and feeding advice) has contributed to significant reductions in diarrhoea associated mortality, 
over half a million children continue to die annually as a result of acute diarrhoeal episodes. In 
addition, there is increasing interest in the rates of mortality in young children in the 90-180 
days following an episode of acute diarrhoea. The long-term benefits of antibiotic 
administration may result from direct antimicrobial effects on pathogens or from other 
incompletely understood mechanisms including improved nutrition, alterations in immune 
tolerance or improved enteric function. Optimizing antibiotic treatment of acute diarrhoea 
episodes in very young children with severe disease may offer the opportunity to significantly 
reduce diarrhoea associated deaths in the 90-180 days following presentation for acute 
diarrhoea and may also improve growth. We propose to evaluate the efficacy of an antibiotic 
(azithromycin) delivered in a specific, targeted fashion to young children (< 2 years of age) at 
high risk of diarrhoea associated mortality in a multi-site randomized, double-blind, placebo-
controlled trial. The study will evaluate the ability of the intervention to reduce mortality within 
180 days of the acute diarrhoeal episode and improve nutritional status over the same period. 
 
 
 

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PROTOCOL VERSIONS AND AMENDMENT HISTORY 
Vers
ion 
Date 
issued Authors  Details of changes made 
WHO ERC submission & 
status; dates and number 
1.0 
05.02.
2016 
Simon, 
Bahl 
Original document, submitted to the WHO ERC 
Submitted 10.02.-16, 
conditionally approved 
10.03.-16 (ERC.0002722) 
2.0 
 
 
Internal working document, not submitted to IRB 
 
3.0 
01.05.
2016 
Simon, 
Bahl 
Provided clarifications based on comments by the 
WHO. No changes to study design 
Submitted 09.05.-16, 
approved 14.07.-16 
(pending site-specific 
protocols) 
4.0 
 
 
Internal working document, not submitted to IRB 
 
5.0 
 
 
Internal working document, not submitted to IRB 
 
6.0 
08.02.
2017 
Ashorn, 
Simon, 
Bahl 
Removed ciprofloxacin from study interventions; 
because of concerns about the safety of this 
antibiotic and a recommendation by a WHO expert 
group. Accordingly, removed “allergy to 
ciprofloxacin” from exclusion criteria, changed 
sample size, and edited analysis strategy to match 
the new 2-arm (vs. earlier 3-arm) design 
Tightened inclusion criteria so that children with 
moderate stunting will not be enrolled, because of 
concerns about spread of antimicrobial resistance 
and recommendation by the WHO expert group: 
Relatedly, clarified objectives and methods for 
biological specimen collection and processing for 
analysis of bacterial resistance to antimicrobial 
agents. 
Added number and cause of hospitalizations in 
outcome measures, based on requests from some 
local IRBs 
Corrected some small errors in the previous version: 
Edited mid upper arm circumference (MUAC) cut-
off to determine severe wasting in exclusion 
criteria, to match WHO and national criteria (from 
MUAC <110 mm to MUAC <115 mm); removed 
“visible wasting” from exclusion criteria (not 
operational); Added “participation in another 
interventional trial” as an exclusion criterion; 
Clarified the definition of some eligibility criteria 
Made some technical edits to the document: Clarified 
the role and meeting modes of Trial Advisory 
Group, Trial Steering Committee, and Data Safety 
and Monitoring Board; Added a cover page with 
list of investigators, a table of contents and a table 
explaining the version history and edits to the 
protocol; Edited informed consent document to 
reflect the changes in the study design; Added an 
informed consent document to be used for a 
guardian interview on the cause of death if a study 
participant dies  
Submitted 08.02.-17, 
clarification sought 
24.02.2017 
7.0 
01.03.
2017 
Ashorn, 
Simon, 
Bahl 
Clarified the storage and analysis of biological 
specimens, edited informed consent on the same. 
Submitted 01.03.-17 
Approved 07.03.2017 
8.0 
30.10.
Ashorn, 
Added a possibility to collect stool samples by rectal 
To be submitted to the 

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Vers
ion 
Date 
issued Authors  Details of changes made 
WHO ERC submission & 
status; dates and number 
2017 
Simon, 
Bahl 
swabs. Added a mother as a possible household 
contact from whom a nasopharyngeal swab can be 
collected for antimicrobial resistance analysis. 
Added a follow-up visit to day 180 for all 
participants. Corrected a typo in participant 
numbers in Figure 1. Edited informed consent on 
changes 
Submitted 02.03.-18 
Approved 25.03.2018 
9.0 
21.12.
2018 
Simon 
Modified Secondary Outcomes 
Modified Analysis Plan based on the secondary 
outcomes changes 
Removed Dr. Per Ashorn as WHO Coordinator  
Adjusted sample size for AMR component 
Submitted to the WHO ERC 
after site approvals of the 
modification 
0. 
BACKGROUND 
The problem 
Despite dramatic reductions in childhood mortality during the past three decades, an estimated 
6.3 million children died in 2013 before reaching their 5th birthday. The reduction in 
mortality has been especially impressive for diarrhoeal disease. In 1980, an estimated 4.6 
million under-five children were dying every year because of diarrhoea (2) while in 2013, the 
number of deaths due to diarrhoea in this age group was reduced to 0.6 million per year (3). 
This very significant reduction was partly due to the diarrhoea control strategy implemented 
throughout the world since the 1980s which includes the treatment/prevention of dehydration 
with ORS and/or IV fluids, administration of zinc, and feeding advice.  
There continue to be over 600,000 deaths/year in under-five children attributed directly to 
diarrhoea. Data from the Global Enterics Multicenter Study (GEMS), a large multi-center 
observational study, suggest that only 1/3 of diarrhoea associated deaths occur in the first 
week following diagnosis. When follow-up visits were conducted 50-90 days after the 
enrolment encounter, the timing of deaths among children with diarrhoea was as follows: 34% 
occurred on days 0–7 after enrolment, 33% on days 8–21 and 33% after day 21 (4). In 
addition, diarrhoea episodes considered as moderate to severe cases had an 8.5fold higher risk 
of death during the 50-90 day follow-up period compared to their matched controls in spite of 
appropriate treatment of the episode, including ORS, zinc and feeding advice (4). The risk of 
death was significantly associated with the degree of stunting at enrolment. Although 26% of 
deaths in cases occurred during the enrolment encounter at the health care facility and 19% 
during a subsequent medical contact, importantly, 55% occurred at home or outside of a 
medical facility. Most deaths in patients with diarrhoea occurred in infants (56%) and toddlers 
(32%). These data suggest that current estimates which predominantly capture immediate 
deaths and those that occur in a health facility may dramatically underestimate diarrhoea 
associated mortality.  
These data also suggest that a proportion of deaths due to diarrhoea, especially in young 
children and in children with moderate to severe diarrhoea, may not be avoided just by using 

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the current treatment recommendations for ORS, zinc and continued feeding. This is 
confirmed by a recently conducted verbal autopsy study which showed that most children 
(70%) who died from diarrhoea had received ORS or intravenous solutions (5) prior to death. 
Additional treatment may be necessary in these children to reduce diarrhoea mortality. 
Potential Solution: 
The World Health Organization currently recommends the use of antibiotics only for the 
treatment of dysentery (as a proxy for suspected shigellosis and cholera) (6, 7). However, 
recent data suggest (4) that multiple bacterial and parasitic pathogens are important in the 
aetiology of moderate to severe diarrhoea and some of these pathogens are significantly 
associated with death in the 90 days following a diarrhoea episode (Cryptosporidium, typical 
enteropathogenic E. coli (tEPEC) and ST-ETEC).   
Given the demonstrated risk of death associated with diarrhoea in specific subsets of children 
with malnutrition and those with severe illness at baseline, expanding the use of antibiotics for 
a subset of children with diarrhoea associated with high risk of subsequent mortality (high 
risk diarrhoea) may be an important intervention to reduce diarrhoea associated mortality.  
Recently published data from the GEMS study suggest that least one putative pathogen can be 
identified in over 80% of children presenting with moderate-severe diarrhoea. In addition to 
viruses (including rotavirus), bacterial pathogens, including Shigella and pathogenic E. coli 
species were commonly identified across all seven of the sites, all with moderate-to-high 
under-5 mortality in sub-Saharan Africa and South Asia (4). Other authors have also 
demonstrated that visible blood in stool is a poor indicator of Shigella infection in children 
with diarrhoea (Pavlinac, ASTMH 2014). Taken together, these data suggest that current 
treatment guidelines may be missing the opportunity to appropriately provide antibiotics to a 
group of young children at particularly high risk of diarrhoea associated mortality. 
Data suggest that 80-90% of all children with diarrhoea currently receive antibiotics, despite 
WHO guidelines restricting antibiotics to a small subset with dysentery or cholera. The use of 
targeted empiric antibiotics for a small subgroup of severely ill children at high risk of death 
may also potentially lower antimicrobial resistance if such children defer to the use of more 
appropriate antibiotics than are commonly used in practice now (despite WHO guidelines) 
and if other children are less likely to use antibiotics based on an assessment of their being at 
lower risk. In addition, data from trials of antibiotics in acute respiratory illness also suggest 
that when clear guidelines are provided for the use of antibiotics in high risk children, 
antibiotic use decreases and resistance among isolated pathogens is reduced (10, 11).  
After discussions with antimicrobial resistance experts at WHO and externally, we feel that 
careful documentation of the association between clinical failure and the development of 
resistance in the community during this trial would be useful to allow careful risk/benefit 
assessment of any proposed policy change if a mortality reduction benefit is shown. In a 
complementary linked study, we propose to study antimicrobial resistance to multiple 
antibiotics, among Escherichia coli and Streptococcus pneumonia isolates in stool samples 
collected from a subgroup of participants at enrolment and in stool samples and naso-

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pharyngeal swabs collected from the same subgroup of participants and their close household 
contacts at the 90-day and 180 days follow-ups. Additionally, we propose that stool be 
collected and stored at -80°C from all participants at enrolment for molecular detection of 
pathogens and identification of antimicrobial resistance genes at a later date. For the same 
purpose, we also propose to store stool and nasopharyngeal swab samples from the subgroup 
of participants at the 90-day and 180-day follow-ups. 
Safety profile of azithromycin: A review of studies having used azithromycin in young 
children concluded that “…azithromycin given as a suspension once daily for 3 days is well 
tolerated and can be used safely for the treatment of acute bacterial infections in children of all 
ages. Even in very young children, there are no concerns regarding safety or tolerance.” (13). 
References 
1. Liu L, Johnson HL, Cousens S, et al. Global, regional, and national causes of child 
mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet, 
2012; 379:2151-2161. 
2. Snyder JD, Merson MH. The magnitude of the global problem of acute diarrhoeal disease: 
a review of active surveillance data. Bulletin of the World Health Organization. 1982; 
60(4):605  
3. GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national 
age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: 
a systematic analysis for the Global Burden of Disease Study 2013. Lancet, 2015 Jan 10; 
385(9963):117-71. (doi: 10.1016/S0140-6736(14)61682-2. Epub 2014 Dec 18). 
4. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, 
Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, 
Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, 
Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, 
Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acácio S, Biswas K, O'Reilly CE, 
Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, Levine MM. 
Burden and aetiology of diarrhoeal disease in infants and young children in developing 
countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. 
Lancet, 2013 Jul 20; 382(9888):209-22. (doi: 10.1016/S0140-6736(13)60844-2. Epub 
2013 May 14..-13). 
5. Rahman AE, Moinuddin M, Molla M, Worku A, Hurt L, Kirkwood B, Mohan SB, 
Mazumder S, Bhutta Z, Raza F, Mrema S, Masanja H, Kadobera D, Waiswa P, Bahl R, 
Zangenberg M, Muhe L; Persistent Diarrhoea Research Group. Childhood diarrhoeal 
deaths in seven low- and middle-income countries. Bull of the World Health Organization. 
2014 Sep 1; 92(9):664-71. (doi: 0.2471/BLT.13.134809. Epub 2014 Jun 23). 
6. WHO. The treatment of diarrhoea: A manual for physicians and senior health workers. 
WHO – Geneva 2005 

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7. WHO. Guidelines for the treatment of Shigellosis. WHO – Geneva 2005 
8. Bardhan PK, Beltinger J, Beltinger RW, Hossain A, Mahalanabis D, Gyr K. Screening of 
patients with acute infectious diarrhoea: evaluation of clinical features, faecal microscopy, 
and faecal occult blood testing. Scandivian Journal of Gastroenterology. 2000 Jan; 
35(1):54-60. 
9. Rogawski ET, Meshnick SR, Becker-Dreps S, Adair LS, Sandler RS, Sarkar R, Kattula D, 
Ward HD, Kang G, Westreich DJ. Reduction in diarrhoeal rates through interventions that 
prevent unnecessary antibiotic exposure early in life in an observational birth cohort. 
Journal of Epidemiology and Community Health. 2015 Nov 30. pii: jech-2015-206635. 
doi: 10.1136/jech-2015-206635 
10. Gouws E, Bryce J, Habicht JP, Amaral J, Pariyo G, Schellenberg JA, Fontaine O. 
Improving antimicrobial use among health workers in first-level facilities: results from the 
multi-country evaluation of the Integrated Management of Childhood Illness strategy. 
Bulletin of the World Health Organization. 2004 Jul; 82(7):509-15. 
11. Qazi SA, Rehman GN, Khan MA. Standard management of acute respiratory infections in 
a children's hospital in Pakistan: impact on antibiotic use and case fatality. Bulletin of the 
World Health Organization. 1996; 74(5):501-7. 
12. Sánchez R1, Fernández-Baca V, Díaz MD, Muñoz P, Rodríguez-Créixems M, Bouza E. 
Evolution of susceptibilities of Campylobacter spp. to quinolones and macrolides. 
Antimicrobial Agents and Chemotherapy. 1994 Sep; 38(9):1879-82. 
13. Treadway G. and Pontani D. Paediatric safety of azithromycin: worldwide experience. 
Journal of Antimicrobial Chemotherapy. 1996; 37, Suppl. C, 143-149 
 
1. 
OBJECTIVES 
1.1 
Primary Objectives: 
1. To compare rates of all-cause mortality in the 180 days following enrolment for an episode 
of high risk diarrhoea without dysentery among children 2 to 23 months of age living in 
low resource settings who are randomized to receive a 3-day course of azithromycin or 
placebo. 
2. To compare the change in linear growth (∆LAZ) in the 90 days following enrolment for an 
episode of high risk diarrhoea without dysentery among children 2 to 23 months of age 
living in low resource settings who are randomized to receive a 3-day course of 
azithromycin or placebo. 

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1.2 
Secondary Objectives:  
1. To compare changes in markers of acute malnutrition (∆MUAC and ∆WLZ and ∆weight) 
in the 90 days following an episode of high risk diarrhoea without dysentery among 
children 2 to 23 months of age living in low resource settings who are randomized to 
receive a 3-day course of azithromycin or placebo. 
2. To evaluate (as determined by verbal and social autopsy- VASA) cause-specific mortality 
rates across randomization arms in the 180 days following an episode of high risk diarrhoea 
without dysentery among children 2 to 23 months of age living in low resource settings 
who are randomized to receive a 3-day course of azithromycin or placebo. 
3.  To compare the proportion of children hospitalized in the 90 days following enrolment for 
an episode of high risk diarrhoea without dysentery among children 2 to 23 months of age 
living in low resource settings who are randomized to receive a 3-day course of 
azithromycin or placebo. 
4. To compare the proportion of children hospitalized or died in the 90 days following 
enrolment for an episode of high risk diarrhoea without dysentery among children 2 to 23 
months of age living in low resource settings who are randomized to receive a 3-day course 
of azithromycin or placebo. 
5.  To compare the proportion of children hospitalized or died in the 10 days following 
enrolment for an episode of high risk diarrhoea without dysentery among children 2 to 23 
months of age living in low resource settings who are randomized to receive a 3-day course 
of azithromycin or placebo. 
6. To compare the proportion of strains of Escherichia coli (E. coli), isolated from stools, 
resistant to selected antibiotics at enrolment among the study population. Since children 
entering later into the trial are coming from the same communities, they can serve as a 
comparator group indicative of the community level of resistance. 
7. To compare the proportion of strains of E. coli, isolated from stools, and Streptococcus 
pneumoniae (S. pneumoniae), isolated from nasopharyngeal swabs, resistant to selected 
antibiotics at DAY 90 and DAY 180 in a randomly selected sub-sample of children enrolled 
in the study and their siblings or close household contacts (children under five years of age 
living in the same household under the care of the same primary caregiver). 
 
2. 
SIGNIFICANCE AND IMPLICATIONS 
Among specifically targeted children at high-risk of diarrhoea-associated mortality we propose 
to determine the efficacy of an antibiotic (azithromycin), compared to placebo, in reducing risk 
of death and linear growth faltering in the six months following an episode of severe diarrhoea. 
Secondarily, we will evaluate the risk of individual and community-level antibiotic resistance 
in bacterial enteric and nasopharyngeal infections. These aims will inform future diarrhoea case 

ABCD trial protocol, version 9.0, 21 December, 2018 
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management guidelines by empirically determining the potential benefits and harms associated 
with targeted antibiotic use in children with severe diarrhoea.  
2.1 
Study sites 
The study will be conducted in health facilities in 7 countries in South Asia (Bangladesh, India 
and Pakistan) and Sub-Saharan Africa (Kenya, Malawi, Mali and Tanzania). The countries have 
been selected based on study site characteristics as well as the strengths and experience of the 
country team in conducting large intervention trials. Within each country, 2-10 individual health 
facilities will be the sites of patient enrolment.   
2.2 
Study Design 
We propose a randomized, multi-country, multi-site, double-blind, placebo-controlled clinical 
trial. A total of 11,500 children (5,750 per treatment arm) will be enrolled in the study across 
the 7 countries and followed up for 180 days (Figure 1). 
Figure 1. Trial design  
 
2.3 
Eligibility criteria 
2.3.1 Inclusion criteria (all of): 
• Children aged 2 – 23 months, presenting to a designated health care facility at a 
participating study site WITH 
• Diarrhoea per caregiver perception AND at least 3 loose or watery stools in the 
previous 24 hours, 
• Diarrhoea for less than 14 days prior to screening AND with at least one of the 
following criteria at presentation: 
A. Signs of some or severe dehydration as per WHO Pocket Book 2013 

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B. Moderately wasted as defined by a mid-upper arm circumference (MUAC) 
less than 125 mm (but greater than or equal to 115 mm) or a weight-for-
length z-score (WLZ) greater than-3SD and less than or equal to -2SD after 
rehydration during stabilization period or  
C. Severely stunted (length-for-age z-score (LAZ) <-3 SD) AND 
• Parent or guardian (caregiver) willing to allow household visits on DAY 2 and 
DAY 3 and willing to return to facility on DAY 90 AND 
• Parent or guardian (caregiver) provides a consent for trial participation on behalf 
of the child, based on local standards 
Exclusion criteria (any one of): 
• Dysentery (gross blood in stool reported by caregiver or observed by healthcare 
worker [HCW]), 
• Suspected Vibrio cholerae infection (determined according to WHO guidelines 
or clinical suspicion), 
• Previously or currently enrolled in the ABCD study, 
• Concurrently enrolled in another interventional clinical trial, 
• Sibling or other child in the household enrolled in the ABCD study and currently 
taking study medication, 
• Signs of associated infections (pneumonia, severe febrile illness, meningitis, 
mastoiditis or acute ear infection) requiring antibiotic treatment, 
• Documented antibiotic use in the 14 days prior to screening (not including 
standard use of prophylactic antibiotics, i.e. co-trimoxazole use in HIV-exposed 
children), 
• Documented use of metronidazole within the last 14-days, 
• Known allergy or contraindication to azithromycin antibiotics, 
• Severe acute malnutrition (SAM) defined as WLZ less than −3 SD, or MUAC 
less than 115 mm, or edema of both feet, OR 
• Living too far from the enrolment health center to ensure adequate Directly 
Observed Therapy (DOT) on DAY 2 and DAY3 
2.4 
Intervention 
 3-day course of azithromycin or placebo 
2.5 
Duration of follow-up 
180 days for the main study. For subgroup of 1600- 1800 participants, there will also be a day 
180 follow-up visit to collect biological specimens. 
 

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3. 
RATIONALE FOR THE DEFINITION OF STUDY POPULATION AND 
ELIGIBILITY CRITERIA 
3.1 
Study Population 
This age group (2-23 months) corresponds to a standard age-group defined in IMNCI and used 
globally to define treatment recommendations. In addition, diarrhoea associated mortality has 
been demonstrated to be highest in children less than 2 years of age especially in malnourished 
children. 
 
3.2 
Inclusion criteria 
Children aged 2-23 months presenting to health facilities for diarrhoea will be screened for 
enrolment. For this trial, we are seeking to enroll young children at high risk of mortality in the 
180 days following an acute diarrhoea episode. This includes children with relatively severe 
diarrhoea. Since there is no standard definition used in clinical practice for assessment of 
severity of all diarrhoea in these settings, we propose using signs and symptoms that are usually 
agreed upon as criteria and objective signs associated with increased risk of death and are also 
used and understood by health care workers. These signs include those of “some” or “severe” 
dehydration as per the 2013 WHO Pocket Book for Hospital Care for Children which are 
established to select the tertile with most severe diarrhoea. In addition, children with evidence 
of malnutrition (severe stunting or moderate wasting) are at high risk of diarrhoea associated 
death (associated with as many as 11% of deaths). 
3.3 
Exclusion criteria 
Per current WHO guidelines, children with dysentery, suspected cholera, severe acute 
malnutrition (SAM), and/or other signs of infections requiring antibiotics should receive 
antibiotic therapy and therefore, could not be ethically randomized to the placebo arm. As a 
result, children presenting with a history of bloody stool (per caregiver report or HCW 
observation), children in a location with a declared cholera outbreak and presenting with 
presumed cholera, children with SAM, and/or children with signs of associated infections, will 
be excluded. Associated infections will be suspected if a child has signs of pneumonia/severe 
pneumonia (cough or difficulty breathing with any one of the following signs: general danger 
signs as per WHO guidelines, chest in-drawing, or fast breathing), severe febrile illness (or 
temperature > 38.0°C with either danger signs or stiff neck), mastoiditis or acute ear infection 
(ear problem with any of the following signs: tender swelling behind the ear, pus draining from 
the ear and discharge reported for less than 14 days, or ear pain).  
 

ABCD trial protocol, version 9.0, 21 December, 2018 
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4. 
RECRUITMENT 
Participants will be identified and recruited by trained study staff from outpatient and inpatient 
departments of study health facilities. Site specific recruitment plans are detailed in each site 
implementation strategy document (see attached). 
4.1 
Screening 
After a potentially eligible child has been identified, the study staff will screen the child for 
eligibility based on the above-mentioned inclusion and exclusion criteria using a standardized 
screening form (see Core Variable Table). If the child presents with diarrhoea and “no signs” 
of dehydration, screening will be continued immediately (anthropometric measurements, 
physical examination, etc.) and the child enrolled if eligible. 
4.2 
Stabilization period  
If the child has signs of “some” or “severe” dehydration, or if the child requires urgent care or 
the screening physician is unsure of the diagnosis, the child will be kept under observation. 
During this “stabilization” period, oral and/or IV rehydration will be conducted, and treatment 
of all urgent conditions will be performed using standard treatment in accordance with the 
WHO Pocket Book of Hospital Care for Children, 2013. If rehydration of the child is 
successfully completed, as well as urgent care provided and diagnosis confirmed, screening will 
be continued (anthropometric measurements, physical examination, etc.) and the child enrolled 
if eligible. However, if the child is not stabilized or requires additional treatment, the child will 
not be screened further. The child will be eligible again if stabilized at a later stage, but in this 
case the screening process will be started all over again.  
4.3 
Consent 
After the child has been found to be eligible to be enrolled in the study, the accompanying 
primary caregiver will undergo informed consent in his/her local language. During consent, 
purpose of the study as well as all study procedures will be explained to the caregiver including 
the administration of the first dose of the study drug by study staff, follow-up visits by study 
staff to give the second and third doses, and collection of stool and nasopharyngeal samples 
(see attached Generic Informed Consent Form). 
4.4 
Enrolment 
After the caregiver has agreed to let the child participate in the study and signed the Informed 
Consent Form, the child will be enrolled and given an enrolment number that will determine 
the study treatment that this child will receive.  
We expect to enroll approximately 105 children per week from all sites combined to reach a 
target enrolment of 11,500 children within the study period. Individual sites will be allowed to 
enroll up to 2000 study subjects until the total sample size is achieved.  
 

ABCD trial protocol, version 9.0, 21 December, 2018 
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5. 
STANDARD MANAGEMENT AND INTERVENTION 
Both groups will receive standard of care for diarrhoeal disease, including zinc, rehydration, 
and nutritional counselling following WHO guidelines. Children with some or severe 
dehydration will be rehydrated and stabilized prior to completion of screening.  
As part of standard case management, caregivers will be advised to seek care immediately if 
their child is unable to drink or to breastfeed, develops fever, starts passing blood in the stools 
with continued diarrhoea or becomes sicker. In addition, as per standard WHO guidelines, the 
caregiver will also be advised to bring their child back to the health facility for a follow up at 
DAY 5 if the child’s clinical status is not improving.  
Caregivers will be counseled at enrolment that children with continuing diarrhoea at DAY 5 
should return to an appropriate level health facility for further treatment.  
5.1 
Study Drugs Intervention 
Based on the enrolment number, a packet with the same number label containing the 
intervention bottles for the child will be opened. The packet will contain a study medication 
displaying child’s enrolment number and a specific measuring/dispensing device. These 
containers will be such that they cannot be contaminated with any external substance and yet 
be baby friendly to feed the dose. The exact nature of these containers will be finalized by 
review of options and in consultation with the manufacturer of the drugs. The details will be 
described in the manual of Standard Operating Procedures (SOP).  
The study medication bottle will contain either a 3-day course of azithromycin or placebo and 
will be given as 1 dose per day (10 mg/kg). The first dose of the study drugs will be given at 
the health facility by a trained study worker. At this time, the caregiver will be trained on how 
to administer the study drug at home. On DAY 2 and DAY 3, a study health worker will visit 
the home of all enrolled children, to provide the subsequent doses of the study drugs or to 
observe the caregiver giving it.  
A fecal sample will be collected and stored from all participants at the enrolment visit, to study 
the emergence of bacterial resistance to antimicrobial agents among study participants, their 
household contacts and the community, as described in more detail in section 6.5 below. 
5.2 
Randomization and blinding 
Randomization (1:1) will be done in random sized blocks (with varying block sizes of 4, 6 
and 8). All drug and placebo will be procured by WHO MCA Department where a separate 
randomization list for each of the sites will be generated. This list will then be converted into 
a unique sequential serial number for each enrolled child for each site. The list provided to the 
site will only have these serial numbers with the first letter depicting the site. Individual 
child’s supply of study drugs will be provided to each site and labeled with this serial number. 
At the sites, enrolled children will receive the drugs contained in the bottle with the 
randomization number identical to the enrolment number. The sites will not have the 
randomization code.  

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To ensure blinding, the number of bottles and doses for each of the two groups will be 
identical. The drug and control bottles and medications will be similar in all aspects including 
the content, color and taste. Treatment allocation (once assigned) will remain blinded to the 
participant, the site PI, the study site staff and the hospital clinicians during all data collection 
phases of the study. 
5.3 
Study drugs administration 
5.3.1 First dose of the study medications 
At enrolment the physician/nurse/study worker enrolling the child will administer the first dose 
of the medication assigned to the child according to his enrolment number. In addition to 
administering the medication s/he will educate and demonstrate to the caregiver the method of 
administration as well as the quantity to be administered using the measuring system. This 
training will help the caregiver to administer doses 2 and 3 which will be given at the child’s 
home (by or under direct observation of the study staff).) In addition, s/he will provide to the 
caregiver user-friendly instructions in the local language and pictorially, both for quantity and 
for administration of the assigned drug to be taken at home. In case the child is admitted in the 
facility all the doses of medication will be administered by the study staff as long the child is 
admitted. When the child is sent home the education previously imparted will be reinforced. 
If a child is enrolled in the afternoon, the caregiver will be instructed to give the second daily 
dose in the morning of the following day. After administration of the first dose of the 
medication, the child will be observed for 30 minutes. If vomiting occurs during these 30 
minutes, a repeat dose of the medication will be administered. If this dose also is vomited, a 
second repeat dose will not be given.  
The caregiver will be asked to return to the health facility if any of the following signs appear 
in the next 10 days: 
 - Anaphylactic reaction, Quincke oedema (angioedema), convulsions, cutaneous rash, 
arthralgia, urticaria, and severe colitis. (see section on Serious Adverse Events) 
5.3.2 Other doses of the study drugs 
When the child is sent home (day 1 for outpatient or short admissions and day 2/3 if the child 
stays in hospital longer) a study worker will accompany the caregiver for ascertainment of the 
address and location of the house. H/she will deliver the medication bottle with the measuring 
device to the caregiver at the household and reinforce the message concerning the 
administration of the subsequent doses.  
On Day 2 and Day 3, a study worker will visit the household and observe the caregiver giving 
the medications to the child in his/her presence or to give the study drugs if the guardian 
prefers that. In addition, s/he will inquire about the child’s symptoms and care after 
administration of the previous dose. If the child vomits the medication within 30 minutes of 
the administration of the study medication, a repeat dose will be given and child will be 
observed for another 30 minutes. A third dose will not be given even if the child vomits again. 

ABCD trial protocol, version 9.0, 21 December, 2018 
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On the last dosing day, the study worker will collect the bottle for the completed medication. 
 
6. 
FOLLOW UP 
The follow-up is summarized in Figure 2 below and described in more detail in the subsequent 
sections. 
Figure 2: Follow-up plan 
 
6.1 
Day 45 Follow up 
Non-medically trained study personnel will contact study participants, using a method of “light 
contact” at DAY 45 (range DAY 40 to DAY 50). This will involve contact by phone or in-
person contact. The purpose of this contact is to ascertain the vital status of the enrolled child 
at DAY 45, ascertain any hospital admissions since enrolment, and to remind the caregiver of 
the DAY 90 follow up. No other outcome determination will be performed at this contact. 
6.2 
Day 90 Follow up 
At the DAY 90 follow up a standardized assessment will be performed by an appropriate 
trained staff to ascertain vital status, hospitalizations, and health of the enrolled child, as well 
as his/her nutritional status (weight, length and MUAC). Participants will be contacted by 
phone up to three times within the DAY 80 to DAY 100 period to fix an appointment for this 
DAY 90 follow up visit that will either take place at the health facility (in which case 
transport will be reimbursed to the caregiver), or at the home of the enrolled child. 
Anthropometric measurements will be performed by a team of dedicated personnel, trained 
according to standard procedures (see SOP manual). 
Children enrolled in the AMR sub-study will also be asked to provide a stool sample. In 
addition, a naso-pharyngeal swab will be taken in the same sub-sample of children. These 
samples will be processed and used to study the emergence of bacterial resistance to 
antimicrobial agents, as described in more detail in section 6.5 below. 
If a child is reported to have died at any time or at the DAY 45 contact or at the DAY 90 visit, 
or at any other point during the study follow up period, specially trained study staff will review 
hospital record (if available) and conduct a standardized verbal autopsy interview to ascertain 

ABCD trial protocol, version 9.0, 21 December, 2018 
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the date and cause of death. The verbal autopsy effort will include capturing all relevant 
hospital/facility-based information on a child that dies in a facility. 
6.3 
Day 180 Follow up 
Study personnel will contact study participants by phone or in person on DAY 180. The purpose 
of this contact is to ascertain the vital status of the enrolled child at DAY 180 and to ascertain 
any hospital admissions since day 90 visit. No other outcome determination will be performed 
at this contact. 
An additional follow up contact / visit will be performed for the randomly selected sub-sample 
of enrolled surviving children who have provided a stool and nasopharyngeal swab sample at 
the DAY 180 visit and their household contact/sibling. At this visit, a stool sample and a 
nasopharyngeal swab will be collected and processed as described in detail in section 6.5. 
6.4 Outcome measurements  
• Mortality and date of death to be ascertained at the DAY 45 contact, the DAY 90 and 
DAY 180 visits, or at any other time point during the 180-day study period. Verbal and 
Social Autopsies (VASA) will be attempted for all recorded deaths within the study 
period. As part of the VASA autopsy procedures for deaths that occur in facilities, we 
will collect all available relevant information (diagnostics, treatment and drugs, etc.) 
from the medical record file of the study subject. 
• Hospitalizations (number and categorized causes) between days 0 and 180 
• Anthropometric measurements: length or height, weight and MUAC will be 
measured at enrolment and at the DAY 90 visit.  
• Antimicrobial resistance prevalence of phenotypic resistance to selected antibiotics in 
isolated E. coli (stool sample), and S. pneumoniae (naso-pharyngeal swabs) at 
enrolment, at DAY 90 and at DAY 180 will be measured on a sub-sample. Additionally, 
stool samples will be stored from all children at enrolment, to facilitate later molecular 
identification of diarrhea etiologies, gut microbiota, and antimicrobial resistance genes 
in stool bacteria. For the same purpose, stool and nasopharyngeal swab samples will be 
stored on DAY 90 and DAY 180 from the sub-sample of children (see sections 6.5 and 
6.6 for further details). 
6.5 
Collection and processing of biological specimens  
A fecal sample will be collected from all children at enrolment. This sample will be aliquoted 
and stored at -80oC without further processing, to facilitate later molecular identification and 
analysis of enteric pathogens and their antimicrobial resistance genes.  
A subset of up to 1600 to 1800 ABCD participants (approx. 15%) will be enrolled in a more 
detailed AMR sub-study. Random assignment into the AMR sub-study will be determined by 
the WHO Central Coordinating Office as part of the intervention randomization code, stratified 
by site and intervention arm. Children enrolled in the AMR sub-study will have a stool sample 
collected and processed at baseline, and a stool sample and a naso-pharyngeal swab collected 

ABCD trial protocol, version 9.0, 21 December, 2018 
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and processed for culture and resistance testing at DAY 90 and at DAY 180 after enrolment. In 
addition, a stool sample and a naso-pharyngeal swab will also be collected from a sibling or 
other close household contact (child contact) of the enrolled child (index-child) at DAY 90 and 
DAY 180 180.  
The stool samples are primarily collected from whole stool passed by the study participant into 
a diaper or a container. If, however, the sample is taken at the study clinic (always on enrolment 
visit, sometimes on subsequent visits), and  the child has not passed stool within 2-4 hours of 
the start of the clinic visit, and the primary caregiver gives a permission to the procedure, the 
stool sample may be taken with 2 cotton swabs, quickly inserted into the participant’s rectum 
by a research nurse or physician.  
Although it is determined at study enrolment whether the index child will participate in the 
AMR sub-study, no samples will be collected from the siblings or close household contacts 
before the 90 and 180-day time points. Eligible contacts are the participant’s mother or children 
aged less than 60 months, who have slept in the same household as the enrolled child for 5 of 
the last 7-nights, and who share the same primary caregiver as the index child. If there are 
multiple contacts in the household who meet the eligibility criteria, then the preference for 
inclusion in the “contact study” will be given to the child closest in age and living in the same 
quarter as the index child. The contact will be the same child at DAY 90 and 180 so as to 
maintain the ability to evaluate within-individual change in resistance. 
For the sub-set of children and their contacts enrolled in the AMR sub-study, before freezing 
of the stool sample at -80°C, a small amount of stool will be immediately placed in Cary-Blair 
transport media and shipped to microbiology facilities (maintaining temperatures between 2-
8°C) within 24 hours of collection. All collected nasopharyngeal swabs will be placed in skim 
milk, tryptone, glucose, and glycerin (STGG) transport medium and transported to 
microbiology facilities for culture and AMR testing. 
Upon receipt at the laboratory, stool samples will be plated on MacConkey agar, incubated at 
35°C - 37°C for 24 hours, and examined for colonies consistent with E. coli. Similarly, naso-
pharyneal swabs will be plated onto selective media and incubated at 35°C - 37°C in 5% CO2 
overnight and examined for colonies consistent with S. pneumoniae. When E. coli colonies are 
identified in the stool samples or S. pneumoniae colonies are identified in the naso-pharyngeal 
samples, distinct colonies will be aggregated into a single cryo vial, aliquoted, and a part will 
be stored -80°C for later genotypic characterization. The remaining sample will be used for 
culture-based AMR testing in laboratories at the participating institutions as described below.  
The antibiotic susceptibility of the collected E. coli or S. pneumoniae isolates will be carried 
out with an automated, commercial microdilution method, using azithromycin and 
approximately 20 other commonly used antimicrobial agents. For testing the sensitivity of E. 
coli to azithromycin, we will use thin inert non-porous plastic carrier strips, impregnated with 
a predefined stable antimicrobial gradient of selected antibiotics (so called E-test, each strip 
containing one antibiotic). Both tests will provide minimum inhibitory concentrations (MICs) 
for each tested antibiotic.  

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6.6 
Storage of collected biological specimens  
The following biological specimens are planned to be stored at -80oC and used for molecular 
identification of diarrhea etiologies, microbiota, and antimicrobial resistance genes in bacteria 
and to assess if any of these characteristics is an effect modifier to the intervention effect. 
1. At enrolment: Stool samples from all participants 
2. At day 90 and day 180: Stool samples from a sub-group (approx. 15%) of participants 
and their close household contacts, participating in an antimicrobial resistance (AMR) 
sub-study. 
3. At day 90 and day 180: Naso-pharyngeal swab samples from the same sub-group of 
participants and their close household contacts, participating in the AMR sub-study. 
The possibility of sample collection and storage will be separately asked from the caregivers of 
study participants during the consent process and the caregivers can separately refuse this aspect 
of the trial. If caregiver consent is obtained, the samples will be stored in secured freezers at 
each of the seven participating institutions. The stored samples are planned to be analyzed in 
batches, within four years after all the sample collection for the trial is completed and with up-
to-date methods and at sites that have capacity to do those analyses. Any leftover samples will 
be destroyed at the end of the above-defined storage period. During the storage, only the local 
PIs and researchers designated by them will have access to the samples.  
The study participants or their caregivers can at any point request the discontinuation of storage 
and destruction of samples collected from them, by contacting the Principal Investigator at her 
/ his study site. Additionally, the study team will strictly adhere to all local regulation at each 
study site on details of sample storage and use. For any transfer of samples to other locations 
during the study period, the ABCD team will seek approval from the local IRBs. 
 
7. 
SERIOUS ADVERSE EVENTS 
Serious Adverse Events (SAE) will be recorded from enrolment to DAY 10 (7 days after the 
end of study intervention). Data on mortality and hospitalizations will be collected until DAY 
180. 
At enrolment, caregivers will be told to come back to the health facility if the enrolled child 
present any of the following signs in the following 10 days: 
- 
Anaphylactic reaction, Quincke oedema, convulsions, cutaneous rash, arthralgia, 
urticaria, and severe colitis. 
On DAY 2 and DAY 3 visit for the DOT, the study worker will record any reported SAE. 
In case there is a suspected allergic reaction caused by the study drug or other SAE, the 
participant will not get any further doses of the study drug. In case of a suspected allergic 
reaction, the study data manager at WHO will break the randomization code for that participant, 
to know which product the participant was receiving. The data manager will provide this 

ABCD trial protocol, version 9.0, 21 December, 2018 
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information to clinicians taking care of the participant, so that they can take in into account in 
the medical management and also counsel the participant’s caregivers about any need to avoid 
certain drugs in future.  
 
8. 
TRAINING 
Before the initiation of patient enrolment, a one-week training of trainers (TOT) that will 
include trainers from each site will be conducted under the supervision of the Maternal, 
Newborn, Child and Adolescent Health (MCA) Department of the WHO at one of the study 
sites. This TOT will include standardization of all procedures for screening and enrolment 
(including assessment of dehydration and physical examination), standard case management, 
randomization, 
medication 
dispensing 
and 
administration, 
follow-up 
procedures, 
anthropometry assessment and verbal autopsy interviews. 
The trainers from each site will then conduct similar intensive training of the study staff in their 
own respective site prior to initiation of enrolment. Refresher training sessions will be repeated 
every two months. Records of these training sessions will be kept for examination during 
external monitoring. In case internal monitoring of the study shows inconsistencies or 
deviations in the conduct of the study, training sessions on specific elements of the protocol 
will be conducted as required. 
 
9. 
TIMELINE 
The total study period is estimated at 43 months, from January 1st 2017 to July 31st, 2020. This 
includes approximately 4 months of preparatory time 30 months of active enrolment (starting 
on May 1st, 2017), six months of follow-up after last enrolment, and 3 months for data cleaning, 
analysis and dissemination.  
 
10. DATA ANALYSIS 
An outline of the analysis plan for the primary objectives is presented here.  
Primary analyses will be intent-to-treat (ITT) based on randomization allocation to the 3-day 
course of azithromycin or placebo. For each analysis, azithromycin will be compared to those 
treated with placebo.  
The primary end-points in this trial are proportion of deaths in the 180 days post-enrolment and 
change in length-for-age z-score in the 90 days post-enrolment by study arm   
Deaths will be ascertained by study staff either through family reports, community health 
workers, hospital staff, or determined at follow-up visits. Date will be determined by verbal 
autopsy or medical records in cases of facility deaths.  

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Length will be assessed using a length board at enrolment and at the day 90 follow-up visit by 
study staff trained in anthropometry. Length will be measured twice at each visit, a third time 
if the two repeated measures varied by more than 10%, and the median length used for analysis. 
Length-for-age z-scores will be calculated using the WHO ANTHRO software. Implausible 
length values will be considered on a case-by-case basis within each site and implausible 
lengths set to missing.   
Loss to follow-up will be determined at 180 days after enrolment and defined as non-attendance 
at the 180-day follow-up visit and inability to be contacted after three subsequent attempts. All 
analyses will utilize a complete case analysis other than in time-to-event analyses where 
children who are lost to follow-up will be censored at the date of last contact with the study 
team. All hypothesis testing will utilize an alpha of 0.05 to determine statistical significance.  
10.1 
Primary Objectives 
1. To compare rates of all-cause mortality in the 180 days following enrolment for an 
episode of high risk diarrhoea without dysentery among children 2 to 23 months of age 
living in low resource settings who are randomized to receive a 3-day course of 
azithromycin or placebo. 
Period prevalence of death will be compared between randomly-assigned treatment groups 
using relative risk regression. Mortality will be defined as a binary variable and will be defined 
as any event of death from time of randomization to end of day 180. Time to death will not be 
considered. Censored individuals will be retained in the denominator. For the primary ITT 
analysis, the following model will be used:  
 
𝑬𝑬(𝒀𝒀|𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓 𝒂𝒂𝒂𝒂𝒂𝒂) = 𝒆𝒆(𝜷𝜷𝟎𝟎+𝜷𝜷𝟏𝟏𝑿𝑿𝒂𝒂𝒂𝒂𝒂𝒂) 
where xazm is an indicator variable specifying randomized to the azithromycin group (xazm=1) 
or not (xazm =0). The risk ratio comparing the risk of death in children randomized to 
azithromycin vs. placebo will be determined by eβ. The statistical significance of this 
comparison will be determined by Wald test.  
If the baseline assessment of randomization reveals an imbalance in characteristics between the 
randomization arms, we will evaluate these characteristics as potential confounders in a sub-
analysis secondary to the ITT. The following baseline characteristics will be evaluated for 
balance between randomization arms (median age, stunting prevalence, wasting prevalence, 
prevalence of “some” and “severe” dehydration, site [Bangladesh, India, Kenya, Malawi, Mali, 
Pakistan, Tanzania], hospital admission, and median duration of diarrhoea). Potential baseline 
confounders will be added stepwise in the relative risk model with randomization arm as 
follows: 
𝒆𝒆൫𝜷𝜷𝟎𝟎+𝜷𝜷𝟏𝟏𝑿𝑿𝒂𝒂𝒂𝒂𝒂𝒂+𝜷𝜷𝒊𝒊𝑿𝑿𝒊𝒊൯ 
Where i are the baseline characteristic(s) included in the model. These will be maintained in the model 
if their inclusion in the model changes the hazard ratio by more than 10%. Also, in a sub-

ABCD trial protocol, version 9.0, 21 December, 2018 
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analysis, separate analysis for each site (Bangladesh, India Kenya, Malawi, Mali, Pakistan, 
Tanzania) will be presented to describe differences in treatment effects between sites (no formal 
hypothesis tests within each site stratum will be performed).  
In a per-protocol analysis, also secondary to the ITT analysis, relative risk regression will be fit 
as described above among the subset of children with documented completion of the full course 
of treatment (direct observation of 3 daily doses and) with at-risk-time beginning on date of 
observed third dose.  
2. To compare changes in linear growth (∆LAZ) in the 90 days following enrolment for an 
episode of high risk diarrhoea without dysentery among children 2 to 23 months of age 
living in low resource settings who are randomized to receive a 3-day course of 
azithromycin or placebo. 
Analysis strategy: Linear regression model will be used to compare mean ∆LAZ across the 
treatment groups in surviving children. ∆LAZ will be operationalized as the difference in LAZ 
between the 90-day follow-up visit and LAZ at baseline. For the primary ITT analysis, the 
following model will be used: 
 
where Y=mean ∆LAZ, and xazm is an indicator variable of being randomized to the azithromycin 
group (xazm=1) or not (xazm=0). The mean difference in ∆LAZ in children randomized to 
azithromycin vs. placebo will be determined by (β). The statistical significance of this 
comparison will be determined by independent t-test. 
If the baseline assessment of randomization reveals an imbalance in characteristics between the 
randomization arms, we will evaluate these characteristics as potential confounders in a sub-
analysis secondary to the ITT as described in the mortality time to event analyses. Potential 
baseline confounders will be added stepwise in the linear regression model with randomization 
arms and maintained in the model if inclusion in the model changes the effect estimates 
(difference in means) by more than 10%.  
In a per-protocol analysis, also secondary to the ITT analysis, a linear regression model will be 
fit as described above among the subset of children with documented completion of the full 
course of treatment (direct observation of 3 daily doses).  
10.2 
Secondary Objectives 
1. To compare changes in markers of acute malnutrition (∆MUAC and ∆WLZ and  
∆weight) in the 90 days following an episode of high risk diarrhoea without dysentery 
among children 2 to 23 months of age living in low resource settings who are randomized 
to receive a 3-day course of azithromycin or placebo.  
Linear regression (and associated z-test) will be used to compare mean ∆MUAC and ∆WLZ 
and weight across the treatment groups among surviving children.   

ABCD trial protocol, version 9.0, 21 December, 2018 
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2. To evaluate (as determined by Verbal and Social Autopsy – VASA) cause-specific 
mortality rates across randomization arms in in the 180 days following an episode of high 
risk diarrhoea without dysentery among children 2 to 23 months of age living in low 
resource settings who are randomized to receive a 3-day course of azithromycin or placebo. 
We will conduct Cox regression and K-M survival analyses (as described for aim 1) for time to 
cause-specific mortality (diarrhoea, pneumonia, malnutrition, other) as separate endpoints to 
understand intervention effects on specific causes of death.  
3. To compare the proportion of children hospitalized in the 90 days following enrolment for 
an episode of high risk diarrhoea without dysentery among children 2 to 23 months of age 
living in low resource settings who are randomized to receive a 3-day course of 
azithromycin or placebo. 
4.  To compare the proportion of children hospitalized or dying in the 90 days following 
enrolment for an episode of high risk diarrhoea without dysentery among children 2 to 23 
months of age living in low resource settings who are randomized to receive a 3-day course of 
azithromycin or placebo. 
5.    To compare the proportion of children hospitalized or dying in the initial 10 days following 
enrolment for an episode of high risk diarrhoea without dysentery among children 2 to 23 
months of age living in low resource settings who are randomized to receive a 3-day course of 
azithromycin or placebo. 
 
6.   To compare the presence of strains of E. coli resistant to selected antibiotics at enrolment 
between children in the different treatment arms entering the trial over time. Since children 
entering later into the trial are coming from the same communities, they can serve as a 
comparator group indicative of the community level of resistance. 
Prevalence of resistance to selected antibiotics and (with Wilson binomial 95% confidence 
intervals) will be calculated on all samples collected in Year 1 of the study, in Year 2 of the 
study and Year 3 of the study and compared using a chi-square test to determine the secular 
trend of evolution of antibiotic resistance at each site. 
7. 
To compare the presence of strains of E. coli, isolated from stools, and S. 
pneumococcus, isolated from nasopharyngeal swab, resistant to macrolide, β-lactamase and 
quinolone at DAY 90 and DAY 180 in a randomly selected sub-sample of children enrolled in 
the study and other children under age 5 years living in the same household. 
Prevalence of antibiotic resistance as a function of treatment group and time will be modelled 
using generalized estimating equations (GEE), with a Poisson link and exchangeable 
correlation structure for each antibiotic and each isolate, to account for repeated measurements 
within individuals. The GEE model will include three time-points (baseline, DAY 90 and DAY 
180) for antibiotic resistance data from E. coli isolates and two time-points (DAY 90 and DAY 
180 for S. pneumoniae isolates). Pairwise comparisons by treatment groups (azithromycin vs. 
placebo) at DAY 90 and DAY 180 and between baseline and DAY 180 (among the E. coli 
isolates) will be conducted using Wald tests. A Wald test will also be used to test the hypothesis 

ABCD trial protocol, version 9.0, 21 December, 2018 
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that the magnitude of change in prevalence, between treatment groups, differs at 90 days and 
180 days (effect modification by time). Because multiple hypothesis tests will be performed (4 
Wald tests per each of 8 antibiotics per each of 2 isolates types =56 tests) we will report p-
values adjusted for multiple comparisons using a Bonferroni correction or a similar method.  
For the siblings and close contacts, the same analytical methods will be used. However, no E. 
coli data from baseline will be collected therefore the E. coli GEE antibiotic resistance models 
will contain only two time points (DAY 90 and DAY 180). 
10.3 
Sample size 
For the purposes of sample size determination for the ABCD trial, we have estimated that the 
180-day mortality in the control group will be 2.7%. Assuming this baseline mortality, a relative 
risk in the intervention group of 0.65 (35% reduction in mortality in the intervention group), 
90% power, 95% confidence, assumed loss to follow-up 10%, and 1:1 ratio in the numbers of 
participants in the control and intervention group, the required sample size would be 5,696 per 
group or 11,392 in total. Since there is an initial plan to conduct one interim analysis for safety 
(under Fleming bounds, assuming equally spaced analyses and maintaining total alpha=0.05 
and beta=0.1, and two-sided symmetric bounds), the sample size will be inflated by a factor of 
1.0091. Hence, the total planned sample size will be 5,750 per group and 11,500 in total. 
The estimated overall mortality in the control group is a summary function of earlier data from 
the GEMS study as well as the assumed proportions of children with various risk factors in the 
sample and respective sub-group specific mortality rates. The 60-day mortality rate in children 
under five years of age with moderate to severe diarrhoea or dysentery in the GEMS study 
averaged 2.0% across all sites. Excluding the two low mortality sites in Bangladesh and West 
Bengal (India), this mortality rate was 2.8%. The mortality estimates for children with various 
degrees of stunting or wasting come from GEMS 1a study (which covers children with less 
severe diarrhoea), supported by meta-analyses of some 10 child cohorts, collected between 
1977 and 1997 (Black et at, Lancet Nutrition Series 2008; Olofin et al PLoS One, 2013).  
As the study is implemented in 7 countries, on average each country will need to enroll 1645 
children (about 822 per study group). Over an enrolment period of 30 months, this would mean 
approximately 13 children/country will need to be enrolled per week.  Individual sites will be 
allowed to enroll up to 2000 study subjects until the total sample size is achieved.  
Aim 2 will compare change in length-for-age Z-score (∆LAZ) between the control group and 
intervention from enrolment to 90 days. We have estimated that we will have 80% power to 
detect at least a 0.04 difference in mean ∆LAZ between study groups using a two-sided t-test 
to compare the difference in mean change in LAZ between two groups with α=0.05 and 
standard deviation (SD) of 0.7 in both groups. This is comparable to results observed in the 
Global Enterics Multicenter Study (GEMS). As the SD of the difference in ∆LAZ has been 
                                                             
1 Jennison C, Turnbull BW. Group Sequential Methods with Applications to Clinical Trials. Chapman and 
Hall/CRC,1999 (page 150) 

ABCD trial protocol, version 9.0, 21 December, 2018 
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shown to vary, Table 1 below outlines the minimum detectable difference in ∆LAZ with 
varying SD.  
Table 1. Sample size and minimum detectable difference in ∆LAZ with varying power and 
standard deviation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
We have estimated that we will have 90% power to detect at least a 0.04 difference in mean 
∆LAZ between study groups (5,750 children per group) using a two-sided t-test to compare 
the difference in mean ∆LAZ between two groups with α=0.05 and standard deviation (SD) of 
0.6 in both groups. Assuming 50% of children will be less than 12 months of age at enrolment 
(2,85085 infants per group), we will have 90% power to detect at least a 0.06 difference in 
mean ∆LAZ between study groups comprised of infants and separately, between study groups 
comprised of toddlers (12-23 months). As a point of reference, combined across all sites in 
GEMS, Moderate to Severe Diarrhoea (MSD) cases lost 0.04 more in LAZ, on average, than 
controls among infants (p<0.001) and among toddlers, MSD cases lost 0.10 more in LAZ than 
controls (p<0.001).  
 
For secondary objectives 6-7, the number of children enrolled in the AMR sub-study was 
determined for comparison of azithromycin resistance prevalence between the azithromycin 
and placebo arm on DAY 90 and DAY 180. The analysis is planned to be done regionally 
(separately for Asia and Africa), because we assume similarity in AMR within a region but 
differences between the two regions. Hence, all the numbers below indicate numbers / region. 
The minimum isolate number required per treatment group was calculated assuming an alpha 
of 0.05 for two-way antibiotic resistance prevalence comparisons between randomization arms. 
We assumed a 15-20% prevalence of azithromycin resistance in the placebo arm at DAY 90, a 
slightly more conservative estimate than azithromycin-resistance estimates among Shigella 
isolates in Mirpur Bangladesh (22%) and in E.coli isolates in the Kongwa District of Tanzania 
(19%). We assumed that the increase in AMR would be about 2-fold at 90 days post enrolment. 
This is based on studies comparing azithromycin resistance in S. pneumoniae and E. coli 
between communities with and without community-wide azithromycin distribution which 
found resistance prevalence in the azithromycin-treated communities to be at least two-times 
Scenario 
Minimum 
detectable 
difference in mean 
∆LAZ  
Standard 
deviation  
(equal SD per 
group) 
Sample size (per 
group) 
at 90% power 
Sample size (per 
group)  
at 80% power 
A 
0.06 
0.7 
2862 
2138 
0.6 
2103 
1571 
0.5 
1461 
1092 
B 
0.05 
0.7 
4120 
3078 
0.6 
3028 
2262 
0.5 
2103 
1571 
C 
0.04 
0.7 
6437 
4809 
0.6 
4730 
3533 
0.5 
3285 
2454 

ABCD trial protocol, version 9.0, 21 December, 2018 
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higher at 3 (30% vs. 10%) and 6 (23% vs. 12%) months after the antibiotic administration 
compared to untreated communities. 
To achieve 90% power to detect a two-times higher prevalence in the azithromycin-treated 
arms, compared to 20% prevalence in the comparison group, we will need 119 E. coli and 119 
S. pneumoniae isolates per treatment arm. We set this as the minimum number of children and 
siblings or close household contacts, from whom specific bacterial colonies would be available 
at the last data collection point on day 180. Accounting for losses at various points of study (as 
indicated in Table 2 below), a total of 345 children per arm (or 690 in total) would need to be 
recruited from both study regions (Asia and Africa).  Given all the uncertainties in our 
assumptions and for logistical ease, we agreed to collect up from 1600 to 1800 study subjects 
allowing each site to collect isolates from approximately 250 children to this sub-study.  For 
the four sites in Africa, this translates to approximately 1000 children enrolled in the AMR 
study and approximately 750 for the three sites in Asia 
Table 2. Assumed losses of participants at the various stages of the AMR sub-study 
 
The death or loss to follow-up estimate (10%) is the same as used for the main sample size 
estimation. Refusal of AMR sampling (20%) and non-availability of any sibling (20%) are the 
research groups predictions on the average situation at the various sites and the bacterial 
colonization rates are based on earlier data that S. pneumoniae carriage proportions range from 
65-81% and E.coli grows in approximately 76-91% of stool cultures.  
 
11. 
RISK MITIGATION 
Azithromycin is approved for infants older than 6 months of age and has been widely used 
without significant safety concerns in neonates and infants for a wide-range of bacterial 
infections. Safety and pharmacokinetics have been evaluated in newborn infants with 
gestational ages as low as 24 weeks (13). While several case reports of pyloric stenosis have 
Point in study
Cause of loss
% loss
Number of 
children / arm
Randomization to the AMR 
substudy (day 0)
Done at enrolment
0%
345
Available at day 90 visit
Death, other loss to follow-up 
between days 0 and 90
10%
311
1st sampling of study child (day 
90)
Refusal of AMR sampling
20%
248
Recruitment of sibling or 
household contact (day 90)
No sibling or household 
contact available
20%
199
Identification of specific 
bacteria (day 90)
No colonization with the 
specific bacteria
33%
133
2nd sampling of participant & 
sibling (day 180)
Death, other loss to follow-up
10%
119

ABCD trial protocol, version 9.0, 21 December, 2018 
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been reported in infants receiving azithromycin, the FDA OND Maternal Health Team 
concluded in 2010 that available data do not demonstrate an association between azithromycin 
use and increased risk of pyloric stenosis2. However, to monitor possible incidence of this 
adverse reaction, all study participants will be queried about hospitalizations (for abdominal 
surgery, for diarrhoea and for some other defined causes) at the day 45 and day 90 follow-up 
visits. 
Large-scale use of antibiotics raises concern for the possibility that antimicrobial resistance will 
be induced and ultimately could negatively impact clinical outcomes in the communities in 
which resistance emerges. Several studies have examined the emergence of antibiotic-
resistance among pharyngeal pneumococcal isolates following mass treatment with 
azithromycin (9). Multiple studies have documented relatively rapid emergence of macrolide 
resistance following mass drug administration with azithromycin (9) although rates of 
resistance decline quickly after discontinuation of treatment (12). To monitor possible 
incidence, spread, and disappearance of antimicrobial resistance, a subgroup of participants and 
their siblings or close household contacts will be asked to provide stool and naso-pharyngeal 
swab specimens before, during, and after the trial intervention, as described in more detail in 
section 6.5. 
Current efforts to reduce antimicrobial use are failing and new strategies and approaches to 
change prescribing practices and patient demand factors are urgently required. Given the 
widespread and inappropriate use of antibiotics, efforts to identify and focus use in a sub-
population of children with well-defined and highly specific acute diarrhoea episodes could 
reduce overall use. This dynamic has been observed for other infectious diseases34. The ABCD 
Trial results, if robust, might thus, paradoxically, lead to reduced use of antibiotics and lessen 
the ecologic pressure pushing resistance. To further limit antibiotic use, the ABCD trial 
enrolment criteria have been made as strict as possible, only including children whom a 
guardian brings to a health facility and who has at least moderate dehydration, moderate wasting 
or severe stunting. 
The mortality estimates used to determine the sample size required to achieve adequate power 
and based on a large multi-center observational study (GEMS), a related observational study 
(GEMS1a) and meta-analyses of earlier cohort studies in Sub-Saharan Africa and Southern 
Asia. Although the estimates used are considered conservative based on other available data 
and preference was given to the selection of sites thought to experience high diarrhoea 
associated mortality, it is conceivable that there will be fewer mortality events observed in this 
trial, which would have implications for study power. 
                                                             
2 Eberly MD, Eide MB, Thompson JL, Nylund CM Azithromycin in Early Infancy and Pyloric Stenosis. 
American Academy of Pediatrics. Published Online (date) February 16, 2015, doi: 10.1542/peds.2014-
2026 
3 Qazi, S.A, Rehman, G.H, Khan, M.A. Standard management of acute respiratory infections in a children’s 
hospital in Pakistan: impact on antibiotic use and case fatality. Bull. WHO, 1996, 74 (5): 501-507 
4 Gouws, E. Bryce, J., Habicht, J.P., et. al. Improving antimicrobial use among health workers in first-level 
facilities: results from the Multi-Country Evaluation of the Integrated Management of Childhood Illness 
strategy.  Bull. WHO, 2004 82 (7): 509-515 

ABCD trial protocol, version 9.0, 21 December, 2018 
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12 
STUDY MANAGEMENT AND OVERSIGHT 
12.1 
Coordination 
The WHO coordinators in the MCA will be responsible for the oversight of the trial, including 
harmonization of methods and procedures across the study sites. The MCA, WHO, is 
responsible for developing technical guidelines, including the management of infections in 
under-five-year-old children. MCA has the operational advantages of building on existing 
WHO facilities at the International, Regional and Country level. This includes a 
communications network and a managerial group with experience in successful handling of 
projects of a similar size and complexity. MCA also has a large network of collaborators in 
academic and other institutions around the world, which have different expertise in 
epidemiology, clinical, microbiological, radiological and diagnostic areas of childhood 
illnesses including diarrhoeal disease. The WHO coordinating team will provide technical 
support in the development of site protocols, site-specific study instruments and standard 
operating procedures. The WHO coordinating team will also oversee randomization and will 
be also responsible for procuring the antibiotic and placebo, arranging for their adequate 
packaging into full-treatment courses and delivering the packaged treatments to the study sites.  
The WHO coordinating team will develop guidelines for monitoring and evaluation of the 
progress of the trial and will be responsible for internal and external monitoring and oversight 
of quality assurance procedures. WHO study coordinators and others identified by them will 
ensure that at least two structured monitoring visits are conducted to each site every year.  The 
monitoring visits will have as their primary aim quality control and the improvement of study 
implementation. The monitors will make direct observations of all relevant study procedures 
and data management activities. The content of monitoring will vary in response to the stage of 
study implementation. Monitoring will start with a review of the sites’ readiness to begin 
implementation. After implementation has started, it will shift its focus to the adequacy of 
procedures for recruitment (including informed consent and adherence to enrolment criteria), 
treatment delivery and follow-up. More intensive monitoring visits are planned during the first 
six months of study implementation than in the later part of the study. This process is intended 
to ensure early problem-identification and prompt resolution at the individual site level and the 
identification of any variability of procedures across sites that might require rectification.  
12.2 
Local Governance 
A Principal Investigator (PI) from each site will be the leader of the local research team and 
will be responsible for the execution of the agreed protocol and for the integrity of the data. The 
PI will be responsible for local financial management as well as for obtaining the necessary 
local approvals for conducting such a study. 

ABCD trial protocol, version 9.0, 21 December, 2018 
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12.3 
Ethical Review 
The study protocol and associated documents (consent forms, CRFs, etc.) will be submitted to 
the Ethical Review Committee (ERC) at the WHO and the Institutional Review Boards at each 
participating institution for ethical approval.  
12.4 
Trial Advisory Group and Technical Steering Committee 
The Trial Advisory Group (TAG) will provide advice to the WHO coordinating team. This 
small group of leading experts will provide strategic guidance and assist with independent 
advice in the case of difficult issues in the conduct of the trial. The Technical Steering 
Committee (TSC) will be comprised of the co-PIs from each study site and include members 
from the WHO coordinating team and the Bill & Melinda Gates Foundation. The initial tasks 
for TSC will include assisting in the finalization of the site-specific protocols and study 
instruments. The TAG and TSC will review the progress in the implementation of the study, 
the planning and review of the data analyses and presentation of the study results. WHO will 
facilitate meetings of the TAG at least once per year, whereas the TSG will meet at least 
monthly, normally electronically. 
12.5 
Data Safety and Monitoring Board   
A Data Safety and Monitoring Board (DSMB) has been established by WHO at study initiation 
to monitor severe adverse events (SAEs) and to approve the statistical analysis plan and 
associated stopping rules for benefit, futility, or harm determined using O’Brien-Fleming 
stopping boundaries. This initial meeting will be held before patient accruals are initiated. The 
DSMB includes five members with expertise in clinical trials, statistics, child mortality 
assessment, ethics, and pediatric care in resource limited settings and it is represented by both 
study regions. The DSMB will meet electronically or in person at least every 6 months and an 
in person meeting of the DSMB will occur at least one time per year. Severe adverse events 
(SAEs) related to study participation will be monitored in real-time and will be summarized 
and reported to study investigators, WHO and relevant institutional review boards (IRBs) 
within 48 hours of occurrence. On a monthly basis, frequencies and descriptions of SAEs will 
be pooled by the data management team at WHO and circulated to investigators, DSMB and 
IRBs. When approximately half of the person-time is accrued in the study, the DSMB will 
review an interim data analysis by arm to determine whether stopping boundaries have been 
crossed. 
12.6 
Quality assurance 
 
Field supervisors will be responsible for assuring the training of the field staff is of high quality 
and rigorous. They will schedule the testing and retraining as required at their individual site. 
Assessment of individual study personnel’s abilities to use the standardized enrolment criteria 
and conduct the anthropometric measurements consistently across the study population are key 
responsibilities. They also will be accountable for the site-specific approaches to minimizing 
loss-to-follow-up. These quality assurance approaches will be reinforced by the WHO 
coordinating team during the early site visits.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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Field supervisors will check all forms completed by field workers before data entry. Data 
cleaning quality assurance will be performed using consistency and range checks both at the 
study sites and at the WHO data coordination Centre as described in the data management 
section. Data quality checks will also be applied on a quarterly basis at the WHO and feedback 
will be provided to the Principal Investigators and study managers in all sites.   
PIs will receive brief monthly progress reports from the Data Coordinating Center during the 
entire study period and will participate in regular telephone conferences with WHO staff. The 
monthly progress reports will include the number children assessed, number of children 
recruited, home visits due to be conducted, actual visits conducted, child hospitalizations, 
deaths and VASA conducted. The templates of the monthly progress report will be developed 
by WHO Data Coordination Centre with inputs from the sites. 
The trial will be registered by the WHO as a clinical trial in one or more Primary Registries in 
the WHO Registry Network. 
12.7 
Data management 
The WHO coordinating team, working in collaboration with RTI, will ensure harmonization of 
data collection and data management processes across all sites. All sites will collect information 
on a core set of variables with standard definitions. The WHO will provide a set of range and 
consistency checks that must be applied to these variables, although the exact procedures used 
to carry out these checks will be left to the individual sites. As a general principle, there should 
be maximum flexibility for each of the sites since they have been selected on the basis of their 
capabilities to carry out large trials. 
Each site will be responsible for data entry and initial cleaning of the data, including running 
range and consistency checks as well as periodic reviews of distributions and identification of 
outliers. Each study site will resolve any inconsistencies within their database in consultation 
with their field data collection team and with field verification if needed. Individual sites will 
be required to provide data on the core set of variables in SQL readable format monthly to a 
central data repository established for the project at the WHO. The WHO team will run another 
set of range and consistency checks including checking of consistency of data quality across 
sites quarterly. Any inconsistencies or queries will be notified to the study site, which will be 
expected to check and address the list of queries and resubmit data. Cleaned data from all sites 
will be pooled and stored in a SQL database at the WHO. Data analysis workshops will be held 
in Geneva following completion and unblinding of the trial data. 
The WHO Coordination group will maintain close communication with both the PI`s and the 
Bill & Melinda Gates Foundation assigned Program Officer. Monthly check in calls to review 
progress and challenges will take place between the WHO and BMGF. In addition, BMGF will 
be invited to attend all meetings of the Trial Advisory Group.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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12.8 
References 
1. Liu L, Johnson HL, Cousens S, et al. Global, regional, and national causes of child 
mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet, 
2012; 379:2151-2161. 
2. Snyder JD, Merson MH. The magnitude of the global problem of acute diarrhoeal disease: 
a review of active surveillance data. Bulletin of the World Health Organization. 1982; 
60(4):605  
3. GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national 
age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: 
a systematic analysis for the Global Burden of Disease Study 2013. Lancet, 2015 Jan 10; 
385(9963):117-71. (doi: 10.1016/S0140-6736(14)61682-2. Epub 2014 Dec 18). 
4. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, 
Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, 
Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, 
Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, 
Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acácio S, Biswas K, O'Reilly CE, 
Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, Levine MM. 
Burden and aetiology of diarrhoeal disease in infants and young children in developing 
countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. 
Lancet, 2013 Jul 20; 382(9888):209-22. (doi: 10.1016/S0140-6736(13)60844-2. Epub 
2013 May 14..-13). 
5. Rahman AE, Moinuddin M, Molla M, Worku A, Hurt L, Kirkwood B, Mohan SB, 
Mazumder S, Bhutta Z, Raza F, Mrema S, Masanja H, Kadobera D, Waiswa P, Bahl R, 
Zangenberg M, Muhe L; Persistent Diarrhoea Research Group. Childhood diarrhoeal 
deaths in seven low- and middle-income countries. Bull of the World Health Organization. 
2014 Sep 1; 92(9):664-71. (doi: 0.2471/BLT.13.134809. Epub 2014 Jun 23). 
6. WHO. The treatment of diarrhoea: A manual for physicians and senior health workers. 
WHO – Geneva 2005 
7. WHO. Guidelines for the treatment of Shigellosis. WHO – Geneva 2005 
8. Bardhan PK1, Beltinger J, Beltinger RW, Hossain A, Mahalanabis D, Gyr K. Screening of 
patients with acute infectious diarrhoea: evaluation of clinical features, faecal microscopy, 
and faecal occult blood testing. Scandivian Journal of Gastroenterology. 2000 Jan; 
35(1):54-60. 
9. Rogawski ET, Meshnick SR, Becker-Dreps S, Adair LS, Sandler RS, Sarkar R, Kattula D, 
Ward HD, Kang G, Westreich DJ. Reduction in diarrhoeal rates through interventions that 
prevent unnecessary antibiotic exposure early in life in an observational birth cohort. 

ABCD trial protocol, version 9.0, 21 December, 2018 
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Journal of Epidemiology and Community Health. 2015 Nov 30. pii: jech-2015-206635. 
doi: 10.1136/jech-2015-206635 
10. Gouws E, Bryce J, Habicht JP, Amaral J, Pariyo G, Schellenberg JA, Fontaine O. 
Improving antimicrobial use among health workers in first-level facilities: results from the 
multi-country evaluation of the Integrated Management of Childhood Illness strategy. 
Bulletin of the World Health Organization. 2004 Jul; 82(7):509-15. 
11. Qazi SA, Rehman GN, Khan MA. Standard management of acute respiratory infections in 
a children's hospital in Pakistan: impact on antibiotic use and case fatality. Bulletin of the 
World Health Organization. 1996; 74(5):501-7. 
12. Sánchez R1, Fernández-Baca V, Díaz MD, Muñoz P, Rodríguez-Créixems M, Bouza E. 
Evolution of susceptibilities of Campylobacter spp. to quinolones and macrolides. 
Antimicrobial Agents and  Chemotherapy. 1994 Sep; 38(9):1879-82. 
13.  Sudha Chaudhari, Pradeep Suryawanshi, Shrikant Ambardekar, Manoj Chinchwadkar and 
Arun Kinare. Safety Profile of Ciprofloxacin used for Neonatal Septicemia. Indian 
Pediatrics, 2004; 41:1250.  
14. Jennifer A. Goldman, Gregory L. Kearns. Fluoroquinolone Use in Paediatrics: Focus on 
Safety and Place in Therapy. 18th Expert Committee on the Selection and Use of Essential 
Medicines (2011). 
15. Treadway G. and Pontani D. Paediatric safety of azithromycin: worldwide experience. 
Journal of Antimicrobial Chemotherapy. 1996; 37, Suppl. C, 143-149 
 
13 
SITE IMPLEMENTATION PLANS 
13.1 
Bangladesh 
Study Area 
The study will take place in two hospitals (Dhaka hospital and Mirpur hospital) of the 
International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), situated in Dhaka 
city in Bangladesh. The two hospitals partly cater to the needs of Dhaka city having a population 
of more than 13 million. The Dhaka Hospital of icddr,b is the largest diarrhoeal disease hospital 
in the world. We expect mortality rates up to day 90 of follow up in both the hospitals to be 
higher at around 2%. In Bangladesh, diarrhoea was responsible for almost 20% of total deaths 
in 1980 but 6% of 0.119 million under-five deaths in 2015 [2], even following the treatment 
and prevention of dehydration with oral rehydration salts (ORS) and/or intravenous (IV) fluids, 
administration of zinc, feeding advice, and with antibiotic treatment for cases of bloody 
diarrhoea and cholera [3]. Dhaka Hospital is a 350 bed hospital, provides care and free treatment 
to around 140,000 diarrhoeal patients with or without complications each year. On an average 
250 patients are treated in the hospital each day. However, during the epidemic season 
(observed at least twice a year), the hospital has to treat as many as 1200 patients per day. More 

ABCD trial protocol, version 9.0, 21 December, 2018 
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than 80% of the patients come from poor socio-economic backgrounds from urban and peri-
urban Dhaka. About 42% of the patients fall within the age group of 2-23 months. Among the 
children aged 2-23 months, 35% present with moderate to severe diarrhoea (MSD) and 29% 
with mid-upper arm circumference (MUAC) >115 to <125 mm. The Mirpur hospital of icddr,b 
is a 100 bed hospital treating diarrhoeal patents for the last 5 years and data on proportion of 2-
23 months children, MSD, and MUAC are almost similar as in the Dhaka hospital. Enteric 
pathogens potentially treatable with an antibiotic are common (ETEC [8%], Shigella [6%], and 
Cholera [5%]) at these two urban hospitals [4]. Among Shigella isolates, ciprofloxacin 
azithromycin sensitivity ranges from 60-76% to. However, sensitivity of both the drugs in 
cholera and salmonella was around 100%. 
Recruitment 
Recruitment will begin at the Dhaka and Mirpur hospitals after IRB approval (both WHO and 
icddr,b). A subject enrolment and follow up team (includes physicians, nurses, research 
assistants, health workers, and heath attendants) responsible for enrolled patients at day 1 and 
during subsequent hospitalization (if required) and another mobile team (includes research 
assistants, health workers) focused solely on community sensitization, directly observed 
therapy, and follow-up into the community will be placed at each of the two hospitals.  Study 
staff will work closely with hospital staff to determine when there is a potentially eligible child 
either in the oral rehydration therapy (ORT) corner or in the short stay ward (SSW). The 
children with some or severe dehydration will be treated in the SSW until dehydration is fully 
corrected. Stabilization/rehydration period will not exceed 6 hours before enrolment. The ORT 
corner manages diarrhoeal children without dehydration and those do not require any antibiotics 
and are observed for 2 hours. Children with no dehydration but having moderate malnutrition 
using MUAC (>115 to <125 mm) or having moderate stunting (<-2SD for length for age of the 
WHO median) or moderate wasting (< = 2SD for weight for length of the WHO median) will 
be enrolled from ORT.  Children with MSD will be enrolled from the SSW. The study co-
coordinator (senior medical officer) and study physician will also participate in the daily 
morning health discussion to sensitize caregivers about the study and actively identify 
potentially eligible participants. The follow-up team will be deployed into the community as 
per the protocol and they will decide whether a child needs to seek care at a facility as per 
standard WHO guideline.  
Screening/Enrolment  
After an eligible child has been identified with the screening described above, the study research 
assistant will approach the primary caregiver of the child and describe the study procedures. 
The research assistant will explain that the child and caregiver must be willing to be visited at 
their home for directly observed therapy (DOT) for the next two days, be willing to be visited 
at their home for follow-up 90-days after enrolment, and be willing to provide a stool sample 
from the child at enrolment and at the 90 and 180 day follow ups (for a subset of children). If 
the caregiver is interested in participating, the study staff will obtain informed consent.  
Informed consent will be conducted in local language (Bangla). The parent or guardian (primary 
caregiver) must sign written informed consent (or provide a witnessed thumbprint if not literate) 
prior to enrolment.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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The study physician will interview the primary caregiver of the child to collect required 
information in the case report form. A stool sample will be collected and processed according 
to study protocol. The follow-up research assistant will collect detailed contact information 
from the caregivers using a standardized patient locator form, including drawing of a map of 
home location with the help of the caregiver. We will also ascertain mobile phone information 
of the caregiver for adherence assessment, follow-up visit reminders, and home tracing. If a 
caregiver does not have a personal mobile phone we will obtain the mobile phone information 
of a household member or friend of the caregivers’ choosing. We expect less than 5% of 
participants to be without cell phone access based on our recently accomplished post discharge 
follow-up study in severely malnourished pneumonia children [6]. 
Following consent, each participant will be assigned a unique study Patient Identification 
Number (PID).  A card detailing the PID and the medical personnel responsible for enrolment 
will be given to the primary caregiver of enrolled children. The patient locator form will be the 
only link between the PID and the participant’s name and will be stored in a locked file cabinet 
at each site accessible by only the study physician/nurses and follow-up staff for the purpose of 
patient tracing. Patient enrolment in Dhaka hospital will be done 24 hours a day, 7 days a week 
and in Mirpur hospital from 9 am to 5 pm in each day.  
Standard of Care 
All enrolled children will receive standard of care for diarrhoea management per WHO and 
Bangladesh National Health guidelines.   
In children with acute diarrhoea, this will include: 
a. Standardized assessment of hydration status 
b.  Rehydration and maintenance fluids with intravenous or oral rehydration solutions 
(ORS) as indicated 
c.  Oral zinc therapy for 10-14 days (10 mg for infants < 6 months old; 20 mg for 6 
months and older) 
d.   Recommendation for increased fluid intake and continued feeding at home 
e.  Instructions on when to seek follow-up care. 
Intervention 
Following consent, appropriate acute management, and stool sample collection, the study 
physician will identify the pre-labeled intervention bottle corresponding to the PID. The study 
physician will explain the regimen and the importance of adhering to the intervention. Direct 
observation of first dose of study medication will be performed by study staff at the hospitals. 
In addition, direct observation of the doses on day 2 and day 3 will be conducted by the mobile 
team in the household on day two and day three. At the end of each day at the enrolment centers, 
the assigned health worker of the mobile study team will work with the respective assigned 
research assistant to determine the visitation schedule for subjects enrolled earlier that day and 
walk through the patient locator form.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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Follow-up 
Between 40 and 50 days since enrolment, a follow-up will be conducted to establish contact 
and ascertain vital status. The preferred method of contact for the 45-day follow-up will be 
mobile phone. If no telephone number is provided, or if the participant cannot be reached after 
four attempts, the mobile study team will conduct a home visit to ascertain vital status and 
remind the caregiver of the 90-day follow-up visit. However, if any child is found to have any 
illness during the contact, the child will be encouraged to visit relevant health facilities.  
All enrolled children will be scheduled to be visited at their home for follow-up at 90 days 
(between day 80 and day 100) following enrolment to ascertain vital status and anthropometric 
measurements.  The research assistant will give a phone call reminder one day prior to the 
scheduled visit. During this visit, anthropometric measurements will be obtained by the study 
personnel. Transportation cost will be reimbursed at the follow-up visit thereby incentivizing 
participants to return. If the participant does not return at their scheduled time, study staff will 
attempt to make contact with the primary caregiver via cell phone; if no telephone number is 
provided, or if the participant cannot be reached, study staff will trace the child to the household 
and bring the caregiver and child to the enrolment  center if at any follow-up, a caregiver reports 
that a child has died, study personnel trained in verbal autopsy will review hospital records (if 
available) and will conduct a standardized verbal autopsy at the household to ascertain the 
potential cause of death. 
Data Management 
All data will be collected in real-time using an offline computer-automated questionnaire 
installed on tabs at the sites at each study day.  The database is password-protected and all site 
staff responsible for entering data will have unique usernames and passwords that are controlled 
by the data manager/study statistician in each study sites and will only have rights to enter data. 
The data manager/study statistician evaluates data daily and generates reports on a weekly basis 
to identify any missing or erroneous data. On a monthly basis, the central data manager (WHO 
based) will enable cross-checking of data entered by generating a detailed monthly report. 
Changes to existing data will be tracked using an electronic auditing system and adverse events 
will be recorded in the database. 
Ethical Assurance for Protection of Human rights 
The study protocol and data collection tools, and the informed consent forms to be used in the 
study will be submitted to the Research Review Committee (RRC) and Ethical Review 
Committee (ERC) of icddr,b, and the study would be initiated only after receiving approval of 
the committee. All medical information, description of treatment, and results of the laboratory 
tests performed will be kept under lock and key, only research staff, hospital physicians and 
nurses, and ERC/DSMB members of icddr,b will have access to these information. 
References 
1. National Institute of Population Research and Training (2011) Bangladesh Demographic and 
Health Survey 2011. 
2. UNICEF (2015) Committing to Child Survival: A Promise Renewed. Progress Report 2015. 

ABCD trial protocol, version 9.0, 21 December, 2018 
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3. WHO (2005) The treatment of diarrhoea: a manual for physicians and other senior health 
workers, 4th rev. Geneva; WHO. 
4. Das SK, Ahmed S, Ferdous F, Farzana FD, Chisti MJ, et al. (2013) Etiological diversity of 
diarrhoeal disease in Bangladesh. Journal of infection in developing countries 7: 900-909. 
5. World Health Organization. Chart Booklet: Integrated Management of Childhood Illness. 
Geneva, Switzerland. WHO 2014. 
6. Chisti MJ, Graham SM, Duke T, Ahmed T, Faruque AS, et al. (2014) Post-discharge 
mortality in children with severe malnutrition and pneumonia in Bangladesh. PLoS One 9: 
e107663. 
13.2 
India 
Description of the Study Site 
The study will be undertaken in Meerut district of Western Uttar Pradesh, India covering a 
population of 3,720,000 (~14.6% constitutes the under-five population). District is divided into 
3 Tehsils: Mawana, Meerut and Sardhana and 12 blocks: Kharkhoda, Daurala. Hastinapur, Jani, 
Machhara, Mawana Kala,Meerut, Pariskhitgarh, Rajpura, Rohata, Sarurpur and Sardhana. Both 
public (Department of Health and Family Welfare) and private sector agencies (hospitals, 
nursing homes and clinics) provide health services in Meerut district. The site carries a high 
disease burden in terms of water borne diseases like diarrhoea, dysentery and typhoid, acute 
respiratory illness especially in young children. As per the Annual Health survey of Meerut 
(2011-2012), the incidence of diarrhoea is about 11.7% in children 0-6 yrs (Rural 12.8% and 
urban 9.9%) (upnrhm.gov.in/site-files/IMR_AHS.pdf; Data from the CMO Office, Meerut).  
Overall mortality associated with diarrhoeal diseases is estimated to be 0.13%. The urban poor 
constitute a high-risk group with regard to child health and have a substantially higher 
prevalence of child morbidity and mortality due to malnutrition and infectious diseases. A study 
conducted in the slums of Meerut reported the prevalence of diarrhoea to be as high as 24.2% 
among the under five children (UHRC, 2007-08).  
Study Recruitment Facilities and Catchment Area  
The proposed study will be carried out in two tertiary care hospitals [1 private- Subharti Medical 
College (SMC) and 1 government- Lala Lajpat Rai Memorial (LLRM) Medical College], 1 
district hospital and 9 of the 12 CHCs in rural areas and their catchment area. Diarrhoea 
caseload in these facilities is high as most of the children (meeting criteria for “moderate to 
severe diarrhoea”) get referred to these facilities. As part of the National Rural Health Mission 
(NRHM) ASHA workers have been providing community health services. They will be used 
for referring diarrhoeal cases to the facilities.   
Catchment Area: In rural setting we have identified the villages covering ~1 lakh population to 
define the catchment area under each CHC. In urban setting, the city area around the health 
facilities (~7-10 Kms radius) has been identified as catchment area.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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Community mobilization: Before the start of the study discussions will be held with the 
Director, NRHM and CMO, Meerut district. PI and Co-PI will visit all the selected health 
facilities and explain the study purpose to the health personnel. Village level meetings will be 
held to mobilize the community support and generate awareness.  
Study Team: The study team will have four different levels of study personnel with specific 
roles and responsibilities. The study team will include Project manager, one study coordinator, 
one-supervisor/quality control personnel at SMC, one at LLRM, 1 at District Hospital and one 
at each of the CHCs. They will work closely with the hospital staff to identify and recruit 
children. Additionally mobile study team – 2 members at SMC, 2 at LLRM, 2 at district 
hospital, 3 at CHCs (covering 3 CHCs per person) will be deployed. These members will be 
provided motorbikes and they will be responsible for direct observational therapy and follow 
up visits. In addition there will be Lab manager, data manager, lab staff, and anthropometry and 
quality control team members.  
Screening and Enrolment 
A surveillance system will be set up at SMC, LLRM and District hospital OPD/Emergency 
wards will identify all diarrhoea cases and screen them for eligibility according to study 
procedures. For those children meeting the eligibility criteria, enrolment supervisor will 
approach the parents/caregiver and explain the study procedures to them. H/She will explain 
that the child and caregiver must be willing to be visited at home (or at a pre-determined nearby 
facility) for directly observed therapy (DOT) for the next two days, be willing to return to the 
health facility for the follow-up visit on day-90 after enrolment, and be willing to provide a 
stool sample from the child at enrolment and possibly at 90 days (for a subset of randomly 
selected children). If the caregiver is interested in study participation, informed written consent 
will be obtained in local language (Hindi).  
After obtaining consent, the enrolment supervisor will complete the Enrolment form and record 
the baseline information such as socio-demographic characteristics, breastfeeding and 
vaccination history. A stool sample will be collected in a sterile stool container and processed 
according to study protocol  
Following consent, each participant will be assigned a unique subject identification number.  A 
card will be given to the caregiver of enrolled children providing contact information in case of 
an emergency. Subject identification information will be stored in a locked file cabinet 
accessible by only the project manager and study coordinator for the purpose of patient tracing. 
At the time of discharge contact details including phone number of the caregiver or 
relative/friend (in case personal number not available) will be obtained to send follow-up visit 
reminders and for follow up visits We will also take the help of ASHAs (part of government 
health system) working in the community to locate the houses.  
Standard of Care 
All enrolled children will receive standard of care for diarrhoea management per WHO and 
National Health guidelines. 

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In children with acute diarrhoea, this will include: 
a.  Standardized assessment of hydration status 
b.  Rehydration and maintenance fluids with intravenous or oral rehydration solutions 
(ORS) as indicated 
c.  Oral zinc therapy for 10-14 days (10 mg for infants < 6 months old; 20 mg for 6 
months and older) 
d.  Recommendation for increased fluid intake and continued feeding at home 
e.  Instructions on when to seek follow-up care. 
Delivery of Intervention 
Following enrolment, stool sample collection, and acute management, the study supervisor will 
identify the pre-labeled intervention bottle corresponding to the Subject ID. The supervisor will 
explain the treatment regime and visitation schedule of the mobile team for monitoring the 
intervention dose in the subsequent 2 days and will counsel the caregiver on the importance of 
adhering to the intervention. Study supervisor will monitor the first dose administered to the 
child at the health facility. Enrolled subjects information will be entered into the database in 
real time and follow-up visit schedules will be prepared every evening for visits falling on the 
next day. The program manager along with study supervisor will allocate visits to mobile team 
workers of a particular area. In case the child is still in hospital information will be sent to the 
hospital supervisor. All children enrolled will be visited on Day-2 and 3 by the mobile team to 
observe the dose of intervention given. Outcome information will be recorded in a predesigned 
questionnaire in the netbook. Subjects needing medical attention will be advised to visit health 
facilities. For children still in hospital, hospital supervisor will give the dose. 
Follow-up Visits 
A follow-up contact will be established between day 40 and 50 to ascertain vital status (usually 
a phone call). If in case the contact couldn't be established with the family after four attempts 
the study worker will be advised to visit the household. A standardized questionnaire will be 
used to record the vital status of the child and to remind the caregiver of the day-90 follow-up 
visit at the facility.  
All enrolled children and caregivers will be scheduled to visit the health facility on day-90 
(between day-80 and -100) following enrolment to ascertain vital status and record 
anthropometric measurements. Prior to the scheduled visit i.e. 2 days in advance text message 
reminders will be sent to the mobile phone number provided. During this visit, the study 
supervisors stationed at the facility will take anthropometric measurements and mortality 
information since the last time the child was seen by study staff.   If the participant does not 
return at their scheduled time, study supervisor will attempt to make contact with the caregiver 
via mobile phone; if the contact could not be established study team member will visit the 
participant household and facilitate them to visit the nearest health facility.  

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If at any of the follow-up visits, a caregiver reports that a child has died an appointment for 
verbal autopsy will be set up at home to ascertain the cause of death. The study staff trained in 
verbal autopsy will review the hospital records (if available) and conduct interviews in the local 
language ‘Hindi’ describing the events that preceded the death.  
Quality Control: For overall supervision, two mobile supervisory staff will be recruited to 
perform quality checks.10% among the enrolled households will be selected randomly from the 
visit schedule using a computer algorithm. Quality control team will visit these households to 
check if the required visitation by the mobile community team were conducted as per protocol. 
In 5% households they will collect repeat data, which will be used by the program manager to 
verify the accuracy of data. Random quality controls check of anthropometry data and repeat 
measurement will be done in visits requiring anthropometric measurements. 
Data Management: All data will be electronically captured in real time using laptops in the 
facilities and tablets or netbooks in the community. At the end of each day, all data from various 
facilities and community will be synchronized with the central server. The database will be 
password-protected and all site staff responsible for entering data will have unique usernames 
and passwords controlled by the data manager. The data manager will generate the follow-up 
visit schedules on daily basis for each facility. Additionally, the data manager will evaluate data 
daily and generate reports on a weekly basis to identify any missing information or erroneous 
data. An audit trail will be maintained for recording any changes to the existing data. 
Ethical Review: 
The study protocol and associated research instruments will be submitted to the Subharti 
Medical College Ethical Review Board and Lala Lajpat Rai Memorial College Ethical Review 
Board for approval. 
13.3 
Kenya 
Study Area 
The study will take place in Homa Bay and Migori counties in the Nyanza region of Kenya, 
which covers a population of 1.9 million people living within the two counties (50% of which 
are under age 15) and a total area of 5780km2. Cases of moderate-to-severe diarrhoea (MSD) 
from the GEMS Kenya site in the Demographic Surveillance System (DSS) area of the nearby 
Kisumu County, had a case-fatality rate of 3.5%.1 The current study will be conducted outside 
of the DSS site where we expect mortality rates to be higher, and where we are conducting an 
ongoing diarrhoea surveillance study. Eight district, 12 sub-district, and four missionary 
hospitals serve Homa Bay and Migori County and are the referral centers for dispensaries and 
health centers. Enteric pathogens potentially treatable with an antibiotic are common (ETEC 
[4.4%], EPEC [6.3%], Shigella [5.2%]) at three district hospitals in the region (Homa Bay, 
Migori, and Kisii).2 Over 25% of children presenting with acute diarrhoea have at least one 
bacterial pathogen that may be treatable with an antibiotic (ETEC, EPEC, Shigella, 
Campylobacter, and EAEC), an expected prevalence that is likely underestimated due to 
culture-based methods used for bacteria isolates other than for detection of diarrhoeagenic 
Escherichia coli. Among Shigella isolates identified in this study, ciprofloxacin resistance has 

ABCD trial protocol, version 9.0, 21 December, 2018 
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not been identified while 90.5% were resistant to trimethoprim/sulfamethoxazole, 47.6% to 
ampicillin, and 74.6% to tetracycline.3 The HIV prevalence among children presenting with 
acute diarrhoea is 5.2% and 11.3% of HIV-uninfected children are HIV-exposed. 
The government, through the Ministry of Health, is the main provider of health services in the 
study area. The various health facilities are graded from Level 1 to 5 depending on the services 
offered and lower level facilities refer to higher level facilities depending on illness severity. 
Level 1 facilities offer community-level health education and promotion services provided by 
community health workers. Level 2 Dispensaries and Level 3 Health Centre’s largely provide 
outpatient services and limited health care and laboratory services. Level 4 sub-County and 
Level 5 County hospitals offer both inpatient and outpatient services, and limited laboratory 
and radiological services. 
Private and faith-based health facilities complement the services offered by the government 
clinics at a minimal cost. These facilities generally offer inpatient, outpatient, and laboratory 
services as well as routine immunizations. These facilities collaborate with the department of 
public health in disease surveillance and research activities within the study area. 
Recruitment 
Enrolment will begin at six sub-county hospitals (Awendo, Isebania, Mbita, Ndhiwa, Rongo, 
and Rachuonyo) in Homa Bay and Migori county. Additional sub-country hospitals within 
Homa Bay and Migori counties (Migori, Taranganya, Uriri, Ogwedhe, Matosa, Suba, Rangwe, 
Kendu Bay, and Macalder) may be added if necessary for enrolment numbers. Three permanent 
study staff (a clinical officer, study nurse, follow-up counsellor) will be placed at each of the 
selected hospitals. Study staff will work closely with hospital staff to determine when there is 
a potentially eligible child either on the ward or in the outpatient unit, a system that is currently 
used to recruit children in our ongoing diarrhoea surveillance study. The study counsellor will 
also participate in the daily morning health talk to sensitize caregivers about the study and 
actively identify potentially eligible participants. To further enrich enrolment, a mobile team 
focused on community sensitization, directly observed therapy (DOT), and participant follow-
up will be deployed in the community. Each enrolment centre will be assigned a mobile team 
member who will travel throughout the community via motorbike.  
Screening/Enrolment  
After a potentially eligible child has been identified, the study nurse will screen them for 
eligibility according to study procedures. Study staff will explain that the child and caregiver 
must be willing to be visited at the home (or at a pre-determined nearby location) for directly 
observed therapy (DOT) for the next two days, be willing to return to the health facility 90-days 
after enrolment, and be willing to provide a stool sample from the child at enrolment and 
possibly at 90 and 180 days (for a subset of randomly selected children). If the caregiver is 
interested in participating, informed consent will be obtained. Informed consent will be 
conducted in the language of the respondent’s choosing (English, Kiswahili, Kuria, or Luo). 
The parent or guardian (primary caregiver) must sign written informed consent (or provide a 
witnessed thumbprint if not literate) prior to enrolment.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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If laboratory results (including HIV and malaria status) are not available from the 
hospitalization record, a finger/heel prick will be used to collect a small amount of blood for 
malaria thin/thick smear or rapid diagnostic testing, and HIV testing will be performed as 
indicated by Kenya NASCOP Guidelines (HIV testing should be performed in all children 
presenting to a healthcare facility). Any child newly diagnosed with HIV will be referred to the 
HIV Care Clinics at the study sites for follow-up care and treatment. The study staff will 
interview the primary caregiver of the child to collect information on socio-demographic 
characteristics, breastfeeding and vaccination history, and the HIV status of the primary 
caregiver. A stool sample will be collected in a sterile stool container and processed according 
to the study protocol. The follow-up counsellor will collect detailed contact information from 
the caregivers using a standardized patient locator form, including drawing of a map of home 
location by study staff with the help of the caregiver as well as using Google Maps satellite to 
confirm the drawn map. We will also ascertain mobile phone information of the caregiver for 
follow-up visit reminders and home tracing. If a caregiver does not have a personal mobile 
phone we will obtain the mobile phone information of a household member or friend of the 
caregivers’ choosing. We expect less than 5% of participants to be without cell phone access 
based on our ongoing diarrhoea surveillance study. 
Following consent, each participant will be assigned a unique study Patient Identification 
Number (PID). A card detailing the PID and the medical personnel responsible for enrolment 
will be given to the primary caregiver of enrolled children. The patient locator form will be the 
only link between the PID and the participant’s name and will be stored in a locked file cabinet 
at each site accessible by only the study clinician and follow-up staff for the purpose of patient 
tracing.  
Standard of Care 
All enrolled children will receive standard of care for diarrhoea management per WHO and 
Kenya Ministry of Health guidelines.   
In children with acute diarrhoea, this will include: 
a.  Standardized assessment of hydration status 
b.  Rehydration and maintenance fluids with intravenous or oral rehydration solutions 
(ORS) as indicated 
c.  Oral zinc therapy for 10-14 days (10 mg for infants < 6 months old; 20 mg for 6 
months and older) 
d.   Recommendation for increased fluid intake and continued feeding at home 
e.  Instructions on when to seek follow-up care. 
Intervention 
Following consent, stool sample collection, and acute management, the study nurse will identify 
the pre-labelled intervention bottles corresponding to the PID. The study nurse will explain the 
regimen and plan for direct observation, directly observe administration of the first dose, and 

ABCD trial protocol, version 9.0, 21 December, 2018 
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will provide information-motivation-behaviour (IMB) theory based counselling on the 
importance of adhering to the intervention. Direct observation of first dose of study medication 
will be performed by study staff at the enrolment site. In addition, direct observation of the 
doses on DAY 2 and DAY 3 will be conducted by the mobile team in the household on day two 
and day three. At the end of each day at the enrolment centres, the assigned member of the 
mobile study team will work with the facility-based study team to determine the visitation 
schedule for patients enrolled earlier that day and walk through the patient locator form with 
the counsellor.  
Follow-up 
Between 40 and 50 days since enrolment, a follow-up will be conducted to establish contact 
and ascertain vital status of the enrolled child. The preferred method of contact for the 45-day 
follow-up will be mobile phone. If no telephone number is provided, or if the participant cannot 
be reached after three attempts, the mobile study team will conduct a home visit. A standardized 
script and brief questionnaire will be used to ascertain vital status and to remind the caregiver 
of the DAY 90 follow-up visit.  
All enrolled children and primary caregivers will be scheduled to return to the health facility at 
DAY 90 (between 80 and 100) following enrolment to ascertain vital status and anthropometric 
measurements. Phone call reminders will be sent to the phone number provided 1 day prior to 
the scheduled visit. In addition, study staff will call participants to actively remind them of the 
scheduled visit. During this visit, anthropometric measurements and stool sample will be 
obtained by the study personnel. Transportation cost will be reimbursed at the follow-up visit 
thereby incentivizing participants to return. If the participant does not return at their scheduled 
time, study staff will attempt to make contact with the primary caregiver via cell phone; if no 
telephone number is provided, or if the participant cannot be reached, study staff will trace the 
child to the household and bring the caregiver and child to the enrolment centre. If at any follow-
up, a caregiver reports that a child has died, study staff trained in verbal autopsy will review 
hospital records (if available) and will conduct a standardized verbal autopsy at the household 
to ascertain the cause of death.  
Data Management 
All data will be collected in real-time using an offline computer-automated questionnaire 
installed on netbooks at the sites. At the end of each study day, the site staff will sync the offline 
version of the database with an online secure SQL server. The database is password-protected 
and all site staff responsible for entering data will have unique usernames and passwords that 
are controlled by the data manager in Nairobi and will only have rights to enter data. The 
Nairobi-based data manager evaluates data daily and generates reports on a weekly basis to 
identify any missing or erroneous data. On a monthly basis, the Seattle-based data manager will 
cross-check the data being entered by generating a detailed monthly report. Changes to existing 
data will be tracked using an electronic auditing system and adverse events will be recorded in 
the database. In case the offline electronic data entry system is not working, paper forms will 
be used to collect data and entered in the online database when internet is available.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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Ethical and Regulatory Considerations 
The study will be conducted according to Good Clinical Practice (GCP), the Declaration of 
Helsinki, Institutional Review Boards and local rules and regulations of Kenya. Submission of 
the protocol and any protocol amendments to regulatory agencies will occur in accordance with 
local regulatory requirements. Kenya Medical Research Institute, Scientific and Ethical Review 
Unit (KEMRI-SERU) is the IRB of record for the Kenyan Site. This protocol will be submitted 
to the KEMRI-SERU for review and approval. After approval by KEMRI-SERU, it will be 
submitted to the Expert Committee on Clinical Trials (ECCT) of the Pharmacy and Poisons 
Board (PPB) at the Kenya Ministry of Health for review and approval before the protocol is 
implemented. 
13.4 
Malawi 
Study Area 
Blantyre district in southern Malawi has a population of 1.3 million, one large government 
referral hospital (the Queen Elizabeth Central Hospital, QECH) and 23 government primary 
health centres. These facilities treat between 1000-3000 children with non-bloody diarrhoea 
each year. Hospitalised non-bloody diarrhoea from the established VacSurv surveillance 
programme (examining rotavirus vaccine effectiveness) has 6-week case-fatality of 1.7%. Data 
on overall mortality associated with diarrhoeal diseases is lacking. HIV prevalence among 
children presenting with acute diarrhoea is 7% and 20% of HIV-uninfected children are HIV-
exposed. Stunting occurs in 27% of children with diarrhoea in our surveillance, but in Malawi 
DHS 2010 stunting prevalence was 49%. Surveillance for rotavirus, norovirus and bacterial 
pathogens is ongoing. Enteric pathogens potentially treatable with an antibiotic are common. 
Among rotavirus negative stool specimen presence of bacterial pathogens was common ( 
Campylobacter 18%, Shigella 12%, Salmonella 1.2%, and verotoxin-producing E. coli 1%). A 
substantial proportion of children presenting with acute diarrhoea have at least one bacterial 
pathogen that may be treatable with an antibiotic, an expected prevalence that is likely 
underestimated due to culture-based methods used for bacteria isolates.  
Recruitment 
Recruitment will occur at QECH and four primary health centres – Bangwe, Ndirande, Limbe 
and Zingwangwa. Each site will have a team comprising 1 clinical officer, 2 study nurses and 
3 field workers. Study staff will screen potentially eligible children attending study facilities 
for the treatment of non-bloody diarrhoea. A study nurse will also participate in the daily 
morning health talk to sensitize caregivers about the study and actively identify potentially 
eligible participants. Children will be triaged and facility care provided as per standard IMCI 
guideline, including rehydration, zinc provision, nutritional advice. Families will be encouraged 
to attend for review 5 days later. Children requiring referral will be advised to attend QECH 
and the study team alerted of their scheduled arrival. Transport costs will be supported for 
children needing referral.  
A field team will conduct directly observed therapy for children enrolled in the study, and 
perform 90-day follow-up household visits. Anthropometry will be conducted at the 90 day 
visits and referrals made for under-nutrition if clinically indicated.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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Screening/Enrolment  
After a potentially eligible child has been identified they will be screened for eligibility 
according to study protocol. The study nurse will approach the primary caregiver and describe 
the study procedures and obtain informed consent. Caregiver must be willing to be visited at 
the home (or at a pre-determined nearby location) for directly observed therapy (DOT) for the 
next two days, and at 90 day for follow-up visit or be willing to return to the health facility, and 
must be willing to provide a stool sample from the child at enrolment and at 90 days. If the 
caregiver is interested in participating, study staff will obtain informed consent. Informed 
consent will be conducted in Chichewa in the first instance, or English if preferred by the 
family. The parent or guardian (primary caregiver) must sign written informed consent (or 
provide a witnessed thumbprint if not literate) prior to enrolment. Emergency treatment will be 
provided prior to any consent procedures for children with severe illness or IMCI danger signs 
and consent procedures commenced only after stabilization. 
Rapid diagnostic test for malaria and HIV testing will occur in adherence with national 
guidelines. Any child newly diagnosed with HIV will be referred to the HIV Care Clinics at the 
study sites for follow-up care and treatment. After the clinical examination and while ongoing 
rehydration and care is continuing under review of the study clinician, the study nurse will 
interview the primary caregiver of the child to collect information on socio-demographic 
characteristics, breastfeeding and vaccination history. A stool sample will be collected in a 
sterile stool container and processed according to study protocol.  
Study staff will collect detailed contact information from the caregivers using a standardized 
patient locator form, including drawing of a map of home location by study staff with the help 
of the caregiver as well as confirm the drawn map by geocoding using Blantyre’s ePal 
technology. Mobile phone contact information of the caregiver will be recorded for adherence 
assessment, follow-up visit reminders, and home tracing. If a caregiver does not have a personal 
mobile phone we will obtain the mobile phone information of a household member or friend of 
the caregivers’ choosing. We expect less than 25% of participants to be without cell phone 
access based on our ongoing diarrhoea surveillance study. 
Following consent, each participant will be assigned a unique study Patient Identification 
Number (PID).  A card detailing the PID and the medical personnel responsible for enrolment 
will be given to the primary caregiver of enrolled children, and a sticker with the PID placed 
on the health passport. The patient locator form will be the only link between the PID and the 
participant’s name and will be stored in a locked file cabinet at each site accessible by only the 
study clinician and follow-up staff for the purpose of patient tracing. 
Standard of Care 
All enrolled children will receive standard of care for diarrhoea management per WHO and 
National Health guidelines.   
In children with acute diarrhoea, this will include: 
a.  Standardized assessment of hydration status 

ABCD trial protocol, version 9.0, 21 December, 2018 
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b.  Rehydration and maintenance fluids with intravenous or oral rehydration solutions 
(ORS) as indicated 
c.  Oral zinc therapy for 10-14 days (10 mg for infants < 6 months old; 20 mg for 6 
months and older) 
d.  Recommendation for increased fluid intake and continued feeding at home 
e.  Instructions on when to seek follow-up care.  
Intervention 
Following consent and stool sample collection, the study nurse will identify the pre-labeled 
intervention bottle corresponding to the PID. The study nurse will explain the regimen and plan 
for direct observation, directly observe administration of the first dose, and will provide 
information-motivation-behavior (IMB) theory based counseling on the importance of adhering 
to the intervention. Direct observation of first dose of study medication will be performed by 
study staff at the enrolment site. In addition, direct observation of thedaily doses on day 2 and 
day 3 will be conducted by the mobile field team in the household on day two and day three 
(exact timing will be in coordination with the caregiver by phone if possible). At the end of 
each day at the enrolment centers, the assigned member of the mobile study team will work 
with the facility-based study team to determine the visitation schedule for patients enrolled 
earlier that day and walk through the patient locator form with the counselor.  
Follow-up 
Between 40 and 50 days following enrolment, contact will be made with the caregiver to 
ascertain vital status of the participating child, preferably by telephone. If no telephone number 
is provided, or if the participant cannot be reached after four attempts, the mobile field team 
will conduct a home visit. A standardized script and brief questionnaire will be used to ascertain 
vital status and to remind the caregiver of the 90-day follow-up visit.  
All enrolled children and primary caregivers will be visited at home or scheduled to return to 
the health facility at 90 days (between 80 and 100) following enrolment to ascertain vital status 
and anthropometric measurements and collect stool samples. Text message reminders will be 
sent to the phone number provided 2 and 1 day prior to the scheduled visit. During this visit, 
anthropometric measurements will be obtained by the study personnel. Transportation cost will 
be reimbursed at the follow-up visit. If the participant does not return at their scheduled time, 
study staff will attempt to make contact with the primary caregiver via cell phone; if no 
telephone number is provided, or if the participant cannot be reached, study staff will trace the 
child to the household and bring the caregiver and child to the enrolment center. If at any follow-
up, a caregiver reports that a child has died, study staff trained in verbal autopsy will review 
hospital records (if available) and will conduct a standardized verbal autopsy at the household 
to ascertain the cause of death. 
Data Management 
All data will be collected in real-time using offline electronic data capture installed on tablets 
at the sites. At the end of each study day, the site staff will perform data backup. Data will be 

ABCD trial protocol, version 9.0, 21 December, 2018 
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collected from each site and collated weekly to a central secure SQL server. All backup data 
will be encrypted. The database is password-protected and all site staff responsible for entering 
data will have unique usernames and passwords that are controlled by the data manager and 
will only have rights to enter data or make modifications in real time but not to change 
previously entered and saved data. The data manager evaluates data and generates reports on a 
weekly basis to identify any missing or erroneous data. On a monthly basis, the study 
coordinator will check all data and generate a detailed monthly report, an encrypted version of 
which will be sent electronically to the WHO Geneva. Changes to existing data will be tracked 
using an electronic auditing system and adverse events will be recorded in the database. 
The study protocol and associated research instruments will be submitted to the University of 
Malawi College of Medicine Research Ethics Committee and Liverpool University Ethical 
Review Board for review and approval prior to initiating the study. Study drug will be 
approved by the Malawi Poisons, Medicines and Pharmaceuicals Board. 
13.5 
Mali 
Study area 
Mali (Figure 1) is a land-locked country in West Africa 
(population ~15.3 million) ranked by the United Nations in 
2014 as the 12th least developed country.  Only one third of 
adults achieve literacy.  According to UNICEF (State of the 
World’s Children, 2014), Mali’s has the world’s 8th highest 
under 5 mortality rate (128 per 1000 live births), with 
similarly high mortality rates for neonates (42 per 1000 live 
births) and infants (80 per 1000 live births).  Other national 
indicators that may have particular relevance for diarrhoeal 
diseases in children include access to improved drinking 
water (89% of urban and 53% or rural households) and 
improved sanitation (25% of urban and 14% of rural households).  Use of oral rehydration 
solution (ORS) for diarrhoea is 11%.  Most women (80%) report exclusive breast feeding for 
at least 6 months.  Moderate and severe underweight, stunting, and wasting are present in 19%, 
28%, and 9% of children 0-59 months of age, respectively.  Nutritional factors are estimated to 
contribute to 45% of childhood deaths in Mali.  HIV seroprevalence remains low (~1.2%).  
Proposed Study Sites for the Current Antibiotic Treatment for Diarrhoea Study.  
Figure 1.  Regions of Mali. 

ABCD trial protocol, version 9.0, 21 December, 2018 
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Figure 2.   
Sample size estimates for the current study 
are based on an expected mortality rate of 
3% among the control group.  Although it 
is difficult to predict with precision, data 
based on GEMS (Table 1) and GEMS 1a 
suggest that mortality among children at 
the GEMS sites who are eligible for the 
current study may be lower than 3%. We 
plan to compensate for this in the selection 
of sampling frames for the current study.   
Two sampling frames are proposed for enrolment of an 
estimated 2,150 children: the first includes 2 health 
facilities in urban Bamako and the second includes 2 
health facilities in rural Koulikoro (Figure 1).  In 
Bamako: we will recruit from l’Hôpital Gabriel Touré 
(HGT), the major paediatric tertiary care center in Mali, 
and from the CSREF located in Commune VI (Figure 
2). At HGT, recruitment will be ongoing for the 
Vaccine Impact on Diarrhoea in Africa (VIDA) study 
among children from the two quartiers belonging to our 
DSS.  For the current study, we will recruit eligible 
children who live in the remaining 55 quartiers in Bamako.  Recruiting from health care 
facilities in Bamako but outside of our DSS will offer the advantage of accessing a population 
that has not been subjected to repeated sensitization, household visits, etc. which could result 
in a reduction in mortality (Hawthorne Effect).  
In Koulikoro:  we will select two health centers (a CSRef and one large CSCOM) in rural 
Koulikoro for recruitment.  The rationale for this approach is that in Mali, rates of mortality 
(Table 2) and stunting (Figure 3) among children in rural communities (where the majority of 
children reside) are twice that observed in Bamako.  
 
Setting
Post-neonatal 
mortality
(1-11 months)*
Post-infant, 
under-5 mortality 
(12-59 months)*
Population
Bamako
14
18
~1.8 million
Other urban
16
22
Rural
29
49
Other regions
Sikasso
32
48
3.0 million
Kayes
26
38
2.0 million
Koulikoro
26
38
2.4 million
Segou
29
56
2.3 million
Mopti
27
53
2.0 million
Table 2. Geographic Patterns of Stunting and Diarrhoeal Mortality
in Mali (Mali Demographic Household Survey (MDHS 2012-3)
Table 1. Mortality among MSD cases and their matched 
controls at 7 GEMS sites during the 50-90 day follow-up 
Figure 3.  Stunting (<2 
SD Ht for age) in Mali 

ABCD trial protocol, version 9.0, 21 December, 2018 
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Before the study begins, community sensitization and consent will be obtained.  As is the 
practice for all studies conducted by CVD-Mali investigators, meetings will be convened 
throughout Bamako and Koulikoro to inform the communities about the study.  Religious, 
political, cultural, and administrative leaders will be invited along with the general community 
to hear about the purpose, procedures, risks, and benefits of the study.  After a question and 
answer session, the community will render a decision about their willingness to participate.  
Sensitization and education of health care workers also will be required to establish new 
practices for appropriate use of antibiotics for children with diarrhoea, as data from GEMS 
suggest that antibiotics were given or prescribed for the majority of children with MSD. 
Brief summary of the implementation strategy 
 
Screening and enrolment visit.  We will follow the model we used in GEMS to capture 
children with diarrhoea (Kotloff 2012, 2013).  In brief, study personnel will be situated at the 
intake area of up to 5 participating health centers.  A study registrar will ask the caregiver of all 
children 2-23 months of age whether the child experienced diarrhoea, according to the study 
definition.  Those with diarrhoea will be referred to the study clinician who will explain the 
study to the caregiver.  The study clinician will determine whether the child meets eligibility 
criteria.  If so, and the caregiver is interested in participation, informed consent will be obtained.  
The clinician will perform all protocol-specified procedures at the enrolment visit, including: 
a) measuring height, weight, and MUAC, b) performing a medical history and focused physical 
examination; c) ensuring that each child receives the recommended therapy for diarrhoea 
(including rehydration with ORS or intravenous therapy, as appropriate to the level of 
dehydration, and zinc, according to the IMCI guidelines), d) collecting a stool sample from each 
enrolled child; e) acquiring the randomization assignment for children who meet all eligibility 
criteria and whose primary caregiver provides informed consent; f) administering the first dose 
of study drug/placebo to the child (and all subsequent doses that are indicated while the child 
remains hospitalized, if applicable); and e) ensuring that procedures are followed to enable 
relocation of the child for directly observed therapy and follow-up visits.   
Relocating participants for home follow-up will be performed with the assistance of community 
liaisons. In PERCH, cases of severe and very severe pneumonia who lived throughout Bamako 
were enrolled at HGT.  Community liaisons were identified throughout the city.  These are 
literate, nonmedical personnel who are respected in the community.  A liaison will be called 
from the child’s community to accompany the child home and accompany the team during 
follow-up visits.  In PERCH, this system resulted in a successful 30-day follow-up visit for 
>90% of subjects.   
Directly observed therapy.  DOT will be performed according to the procedures described 
above in section 5.  Serious adverse events will be collected as described in Section 7.   
Follow-up visits.  Field workers will perform protocol-designated follow-up visits as 
described in section 6 above.  If a child has died on or before day 90, arrangements will be made 
for the CVD-Mali DSS team to perform a verbal autopsy.      

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The study protocol and associated research instruments will be submitted to The Centre for 
Vaccine Development Ethical Review Board (Mali) and the Ethical Review Board of the 
Maryland University (USA) for review and approval prior to initiating the study. 
13.6 
Pakistan 
Study Area 
This study will be conducted in 8 contiguous low income communities among which 4 sites 
are located   approximately 20 km outside Karachi where the Aga Khan University’s 
Department of Pediatrics and Child Health has maintained demographic surveillance since 
2003. These communities are coastal settlements, with a total area of 8.1 square miles under 
surveillance.  
Each community has a primary healthcare clinic (PHC) run by staff from the Department of 
Pediatrics and Child Health, Aga Khan University.  These clinics also function as study sites 
for various research projects. They provide free primary health care services between 9am to 
4 pm daily for all children < 5 years of age residing in the demographic surveillance area. 
These centers also provide vaccination services as per the Expanded Program of 
Immunization (EPI) in liaison with the local town health officer who provides the EPI 
vaccines. Children requiring more specialized care are given free transport to a major public 
sector children’s hospital in Karachi. The other two sites are public sector hospitals which are 
located in close proximity of the two AKU PHC. These public hospitals are working in 
collaboration with NGO and they are providing health care services to the low income setting 
communities. 
The population according to our last census in these communities is approximately 306,041 
with children under five forming almost 14% (44,273) of the total population. The annual birth 
cohort of these areas is approximately 10,000 The neonatal mortality rate is 45.2 per 1000 live 
births and the U5MR in these areas is 74.3 per 1000 live births The weighted annual incidence 
of MSD determined from the GEMS study was 24.5 (12.2-36.8), for the 0-11 months age group 
and 16.6 (7.3–25.9) and 2.2 (0.8–3.6) per 100 child years for the 12-23 months and 24-59 
months  age groups respectively.  Almost 25% of the children <5 years living in these 
communities have moderate malnutrition (MUAC <12.5cm). Overall mortality associated with 
diarrhoeal diseases is estimated to be 1.3%. 
The remaining two sites are run by a non-profit organization (Sina Trust Foundation) which 
provides primary healthcare services to less-privileged communities. Sina clinics are placed 
across Karachi and more than 80% of Sina patients are women and children They provide 
services to the patients at their doorstep at a very affordable cost. In total 10 clinics of Sina 
Trust are functioning in various urban slums across Karachi, 2 of which will be part of this trial 
Recruitment 
Children will be enrolled from the Aga Khan and Sina managed primary health centers (PHC) 
and ,nearby local hospitals. Each site will have a study medical physician, research assistant 

ABCD trial protocol, version 9.0, 21 December, 2018 
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and community health workers. Every child aged between 2-24 months coming to the centers 
with diarrhoea will be assessed for eligibility.  
At the local hospitals the physician designated for the study will work closely with the hospital 
staff to identify diarrhoeal children including the outpatient and the admitted children. The 
community health workers will also work closely with the local area physicians who will be 
asked to refer diarrhoea patients to the local primary health care center. 
Screening/Enrolment  
After a potentially eligible child has been identified he/she will be screened for eligibility 
according to study protocol. If found eligible,the study physician will explain all the study 
procedures to the primary caregiver. Study details including randomization, drug therapy, 
chance of receiving placebo, home visits for directly observed therapy (DOT) and follow ups 
at 45 and 90 days will be explained in detail. If the caregiver is willing to participate the consent 
will be taken. Consenting will be conducted in the local language (Urdu, Sindhi). Informed 
consent will be taken in the presence of witnesses. If the caregiver is unable to read and write 
and the physician will explain the consent in the presence of a community health worker who 
is not part of the study. The primary caregiver will sign or provide a thumbprint (if illiterate) 
on the informed consent prior to enrolment. 
A stool sample will be collected and processed according to study procedures. All efforts will 
be made to collect information regarding phone numbers of the caregiver and someone else in 
the household. This will be used for adherence assessment, follow-up visit reminders, and home 
tracing if the family moves during the time the child is enrolled in the study. 
Following consent, each participant will be assigned a unique study Patient Identification 
Number (PID).  A card detailing the PID and the study site will be given to the primary caregiver 
of enrolled children. The patient information will be recorded locally by the Research assistants 
in registers and in the site data base. All registers and CRFs will be stored in a locked file cabinet 
at each site accessible by only the study clinician and follow-up staff for the purpose of patient 
tracing. The database will be password protected. 
Standard of Care 
All enrolled children will receive standard of care for diarrhoea management per WHO and 
National Health guidelines.   
In children with acute diarrhoea, this will include: 
a.  Standardized assessment of hydration status 
b.  Rehydration and maintenance fluids with intravenous or oral rehydration solutions 
(ORS) as indicated 
c.  Oral zinc therapy for 10-14 days (10 mg for infants < 6 months old; 20 mg for 6 
months and older) 
d.  Recommendation for increased fluid intake and continued feeding at home 

ABCD trial protocol, version 9.0, 21 December, 2018 
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e.  Instructions on when to seek follow-up care. 
Intervention 
Following consent and stool sample collection, the study physician will identify the pre-labeled 
intervention bottle corresponding to the PID. The study physician will explain the regimen and 
plan for direct observation, directly observe administration of the first dose, and will provide 
information-motivation-behavior (IMB) theory based counseling on the importance of adhering 
to the intervention. Direct observation of first dose of study medication will be performed by 
study staff at the enrolment site. In addition, direct observation of the doses on day 2 and day 3 
will be conducted by the mobile team (community health works) in the household on day two 
and day three. At the end of each day at the enrolment centers, the assigned member of the 
mobile study team will work with the facility-based study team to determine the visitation 
schedule for patients enrolled earlier that day.  
Follow-up 
Between 40 and 50 days since enrolment, a follow-up will be conducted to establish contact 
and ascertain vital status. The preferred method of contact for the 45-day follow-up will be 
mobile phone. If no telephone number is provided, or if the participant cannot be reached after 
four attempts, the mobile study team will conduct a home visit. A standardized script and brief 
questionnaire will be used to ascertain vital status and to remind the caregiver of the 90-day 
follow-up visit.  
All enrolled children and primary caregivers will be scheduled to return to the health facility at 
90 days (between 80 and 100) following enrolment to ascertain vital status and anthropometric 
measurements.  Text message reminders or call to the phone number provided will be done 2 
and 1 day prior to the scheduled visit. During this visit, anthropometric measurements will be 
obtained by the study. Transportation cost will be reimbursed at the follow-up visit thereby 
incentivizing participants to return. If the participant does not return at their scheduled time, 
study staff will attempt to make contact with the primary caregiver via cell phone; if no 
telephone number is provided, or if the participant cannot be reached, study staff will trace the 
child to the household and bring the caregiver and child to the enrolment center. If the caregiver 
is not willing to travel to the center, the follow up will be conducted at home If at any follow-
up, a caregiver reports that a child has died, study staff trained in verbal autopsy will review 
hospital records (if available) and will conduct a standardized verbal autopsy at the household 
to ascertain the cause of death.  
Data Management 
All data will be collected in real-time using an offline computer-automated questionnaire 
installed on netbooks at the sites. At the end of each study day, the site staff will sync the offline 
version of the database with an online secure SQL server.  The database is password-protected 
and all site staff responsible for entering data will have unique usernames and passwords that 
are controlled by the data manager in Pakistan and will only have rights to enter data. The data 
manager will evaluate data daily and generates reports on a bi-weekly basis to identify any 
missing or erroneous data. Changes to existing data will be tracked using an electronic auditing 
system and adverse events will be recorded in the database. 

ABCD trial protocol, version 9.0, 21 December, 2018 
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The study protocol and associated research instruments will be submitted to The Aga Khan 
University Ethical Review Board for review and approval prior to initiating the study. 
References 
1. Kotloff KL, Nataro JP, Blackwelder WC, et al. Burden and aetiology of diarrhoeal disease 
in infants and young children in developing countries (the Global Enteric Multicenter Study, 
GEMS): a prospective, case-control study. Lancet. 2013;382(9888):209-222. 
2. Pavlinac PB, John-Stewart GC, Naulikha JM, et al. High-risk enteric pathogens associated 
with HIV infection and HIV exposure in Kenyan children with acute diarrhoea. Aids. 
2014;28(15):2287-2296. 
3. Pavlinac PB, Denno DM, John-Stewart GC, Onchiri FM, Naulikha JM, Odundo EA, 
Hulseberg CE, Singa BO, Manhart LE, Walson JL. Accuracy of the Integrated Management 
of Childhood Illness (IMCI) algorithm in identifying culture-confirmed diarrhoeal 
pathogens requiring antibiotic therapy. Abstract No 1591, 63rd American Society Tropical 
Medicine and Hygiene Conference, New Orleans, LA, November 2014 (Oral presentation). 
4. World Health Organization. Chart Booklet: Integrated Management of Childhood Illness. 
Geneva, Switzerland. WHO 2014. 
13.7 
Tanzania 
Study Area 
The study will take place in peri-urban Dar es Salaam, Tanzania, with a total population of 
approximately 4.6 million inhabitants. The burden of diarrhoeal disease is high in Tanzania, 
with an estimated 9% of all deaths in children under age 5 due to diarrhoea 1.  In a recent study 
using data from verbal autopsies as part of the DHS survey covering 222,958 persons 280 
childhood deaths due to diarrhoea were recorded. Forty-eight (60%) of these were due to acute 
watery diarrhoea and 19 (24%) were due to persistent diarrhoea2.  In a study to 
identifydiarrhoeagenic Escherichia coli (DEC) isolated from hospitalized infants and children 
in Dar es Salaam, it was found that 22.9% of the 280 children had DEC, with the majority being 
enteroadherent and enteropathogenic E. coli 3. The effectiveness of IMCI-recommended 
antibiotics was also examined. More than 90% of the DEC and Shigella sp. were resistant to 
co-trimoxazole, but all were sensitive to ciprofloxacin 4. In two recent trials conducted in Dar 
es Salaam, all-cause mortality rate was 2% in 2400 infants born to HIV-negative mothers and 
11% in 2387 infants born to HIV-positive mothers 5. 
Recruitment 
For this large, randomized, clinical trial, subjects will be recruited from two busy outpatient 
clinics (Mbagala Rangi Tatu, Mbagala Roundtable) and one large district hospital (Temeke 
District Hospital). Children with diarrhoea are referred from numerous urban and semi-urban 
health facilities throughout Dar es Salaam. Prior to the study start, Drs. Manji and Duggan have 
visited these three sites and described the rationale and logistics of the trial to the hospital 
leadership and staff. Our preliminary communication with these physicians has confirmed a 

ABCD trial protocol, version 9.0, 21 December, 2018 
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high degree of interest in the study, as well as access to patient treatment areas with dedicated 
space.  
Additional recruitment sites may also be used, depending on patient enrolment rates. These 
include:  
1. Main Site (noted above) - Temeke Distric Hospital,  / Satellite =  Mbagala Rangi Tatu, 
Mbagala Round table Yombo Vituka and Yombo Reli ,Vingunguti, Kiwalani, Taima 
Akudo 
2.  Main Site -  Amana Hospital /  Satellite =, AKC CTC , Mnazi Mmoja ,  Gerezani, TRC 
Dispensary 
3. Main Site -  Mwananyamala Hospital / Satellite = Sinza, Tandale, Magomeni, , Kimara, 
Mwenge 
Research nurses will be placed at each of the three selected sites for recruitment. Study staff 
will work closely with hospital staff to determine when there is a potentially eligible child either 
on the ward or in the outpatient unit, a system that has been successfully used to recruit children 
in our past clinical trials. Additionally, a team focused solely on community sensitization, 
directly observed therapy, and follow-up (mobile team) will be deployed into the community, 
enriching enrolment by working with existing Ministry of Health community health clinics that 
commonly refer patients to one of the enrolment hospitals. Each enrolment center will be 
assigned a mobile team member who will travel to participants’ homes (see below).  
Screening/Enrolment  
After a potentially eligible child has been identified, confirmation of all inclusion and exclusion 
criteria will be performed by the study nurse, and will include physical examination of children 
by study physicians to assess degree of dehydration, as well as to confirm the absence of any 
contraindication to study participation.  These will include any IMCI general danger signs (e.g., 
unable able to drink or breastfeed, convulsions, vomiting, unconscious, stiff neck) or evidence 
of another illness requiring antibiotics (e.g., pneumonia) or other drugs that are contraindicated 
(e.g., coartem).  
If no contraindication to study participation is found, a study nurse will approach the primary 
caregiver of the child and describe the study procedures. Study staff will explain that the child 
and caregiver must be willing to be visited at the home for directly observed therapy (DOT) for 
the next two days, be willing to return to the health facility 90-days after enrolment, and be 
willing to provide a stool from the child at enrolment and possibly at 90 days (for a subset of 
randomly selected children). If the child is eligible and the caregiver interested in participating, 
written informed consent will be obtained in Kiswahili. The parent or guardian (primary 
caregiver) must sign written informed consent (or provide a witnessed thumbprint if not literate) 
prior to enrolment.  
After informed consent is obtained, research procedures will commence. These will include 
tablet-based questionnaires for data concerning history of diarrhoea (duration, severity, 
frequency, accompanying vomiting), baseline demographic, socioeconomic and past medical 
history,  breastfeeding and vaccination history, and the HIV status of the primary caregiver and 
child (if known) will be recorded. A stool sample will be collected in a sterile stool container 
and processed according to study procedures.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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The follow-up research nurse will collect detailed contact information from the caregivers using 
a standardized patient locator form, including at least two working cell phone numbers for the 
caregiver, as well as a physical description of the whereabouts of the child’s home (so-called 
“mapcues”). If a caregiver does not have a personal mobile phone we will obtain the mobile 
phone information of a household member or friend of the caregivers’ choosing. We expect 
fewer than 5% of participants to be without cell phone access based on our past diarrhoea 
studies. 
Standard of Care 
All enrolled children will receive standard of care for diarrhoea management per WHO and 
Tanzanian Ministry of Health guidelines.   
In children with acute diarrhoea, this will include: 
a.  Standardized assessment of hydration status 
b.  Rehydration and maintenance fluids with intravenous or oral rehydration solutions 
(ORS) as indicated 
c.  Oral zinc therapy for 10-14 days (10 mg for infants < 6 months old; 20 mg for 6 
months and older) 
d.  Recommendation for increased fluid intake and continued feeding at home 
e.  Instructions on when to seek follow-up care. 
Intervention 
Following consent and stool sample collection, the study nurse will identify the pre-labeled 
intervention bottle corresponding to the patient ID number. The study nurse will explain the 
regimen and plan for direct observation, directly observe administration of the first dose, and 
will provide information-motivation-behavior (IMB) theory based counseling on the 
importance of adhering to the intervention. Direct observation of first dose of study medication 
will be performed by study staff at the enrolment site. In addition, direct observation of the 
doses on day 2 and day 3 will be conducted by the mobile team in the household on day two 
and day three. At the end of each day at the enrolment centers, the assigned member of the 
mobile study team will work with the facility-based study team to determine the visitation 
schedule for patients enrolled earlier that day and walk through the patient locator form with 
the counselor.  
Follow-up 
Between 40 and 50 days since enrolment, a follow-up will be conducted to establish contact 
and ascertain vital status. The preferred method of contact for the 45-day follow-up will be 
mobile phone. If no telephone number is provided, or if the participant cannot be reached after 
four attempts, the mobile study team will conduct a home visit. A standardized script and brief 
questionnaire will be used to ascertain vital status and to remind the caregiver of the 90-day 
follow-up visit.  

ABCD trial protocol, version 9.0, 21 December, 2018 
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All enrolled children and primary caregivers will be scheduled to return to the health facility at 
90 days (between 80 and 100) following enrolment to ascertain vital status and obtain 
anthropometric measurements.  Text message reminders will be sent to the caregiver’s phone 
number 2 and 1 days prior to the scheduled visit. During this visit, anthropometric 
measurements will be obtained by a research nurse, and caregivers will be asked about any 
hospitalizations or other severe adverse event (SAE) occurring since the last time the child was 
seen by study staff. Transportation cost will be reimbursed at the follow-up visit thereby 
incentivizing participants to return. If the participant does not return at their scheduled time, 
study staff will attempt to make contact with the primary caregiver via cell phone; if no 
telephone number is provided, or if the participant cannot be reached, study staff will trace the 
child to the household and bring the caregiver and child to the enrolment hospital. If at any 
follow-up, a caregiver reports that a child has died, study staff trained in verbal autopsy will 
review hospital records (if available) and will conduct a standardized verbal autopsy at the 
household to ascertain the cause of death.  
Data Management 
All data will be collected in real-time using an offline computer-automated questionnaire 
installed on netbooks at the sites. At the end of each study day, the site staff will sync the offline 
version of the database with an online secure SQL server.  The database is password-protected 
and all site staff responsible for entering data will have unique usernames and passwords that 
are controlled by the data manager and will only have rights to enter data. The MUHAS-based 
data manager evaluates data daily and generates reports on a weekly basis to identify any 
missing or erroneous data. On a monthly basis, the Geneva-based data manager will cross-check 
the data being entered by generating a detailed monthly report. Changes to existing data will be 
tracked using an electronic auditing system and adverse events will be recorded in the database. 
The study protocol and associated research instruments have been submitted to Muhimbili 
University Ethical Review Board (Tanzania) and the IRB of Boston Children's Hospital for 
review. 
References 
1. Liu L, Johnson HL, Cousens S, et al. Global, regional, and national causes of child mortality: 
an updated systematic analysis for 2010 with time trends since 2000. The Lancet. 
2012;379:2151-2161. 
2. Rahman AE, Moinuddin M, Molla M, et al. Childhood diarrhoeal deaths in seven low- and 
middle-income countries. Bull World Health Organ. 2014;92(9):664-671. 
3. Moyo S, Maselle S, Matee M, Langeland N, Mylvaganam H. Identification of 
diarrhoeagenic Escherichia coli isolated from infants and children in Dar es Salaam, 
Tanzania. BMC Infectious Diseases. 2007;7(1):92. 
4. Moyo SJ, Gro N, Matee MI, et al. Age specific aetiological agents of diarrhoea in 
hospitalized children aged less than five years in Dar es Salaam, Tanzania. BMC Pediatr. 
2011;11:19. 

ABCD trial protocol, version 9.0, 21 December, 2018 
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5. Duggan C, Manji KP, Kupka R, et al. Multiple micronutrient supplementation in Tanzanian 
infants born to HIV-infected mothers: a randomized, double-blind, placebo-controlled 
clinical trial. Am J Clin Nutr. 2012;96(6):1437-1446.