Improved Polyunsaturated Ready-to-use Therapeutic Food for Improved Neurocognitive Outcomes in Severe Acute Malnutrition
Phase: NA
Conditions: Severe Acute Malnutrition
Sponsor: Washington University School of Medicine

 
Cover Page for Trial Protocol 
OFFICIAL TITLE 
Improved Polyunsaturated Ready-to-use Therapeutic Food for 
Improved Neurocognitive Outcomes in Severe Acute 
Malnutrition 
NCT NUMBER 
NCT03094247 
TRIAL PRINCIPAL 
INVESTIGATOR 
Mark Manary, MD 
Helene B Roberson Professor of Pediatrics, Washington 
University in St. Louis 
INVESTIGATORS 
Kenneth Maleta MBBS 
Department of Community Health, College of Medicine, 
University of Malawi 
 
J Thomas Brenna PhD 
Department of Pediatrics, Dell Medical School, 
University of Texas at Austin 
 
Richard Lane Canfield PhD 
Department of Psychology, Cornell University 
INSTITUTIONS 
Washington University in St. Louis 
University of Texas, Austin 
College of Medicine, University of Malawi 
Cornell University 
FUNDING 
Unorthodox Philanthropy 
Open Philanthropy 
DOCUMENT DATE 
16 July 2021 
 
 
The funding agencies have played no role in the design or conduct of this study and do not 
retain any ownership of the data or other materials collected during the study. There shall 
also be no input from the funders about the data analysis, manuscript, or decision to publish 
any data. All responsibility for the trial design, methods, data analysis, conclusions, and 
decisions regarding publication rest ultimately with Drs. Manary, Maleta and Brenna. 
 
 
 
 
 
 
 
 
 
 

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1 Table of Contents 
1 
Table of Contents 
2 
2 
Abbreviations and Definitions 
4 
3 
Trial Summary 
5 
3.1 
Type of study 
5 
3.2 
Problem to be studied 
5 
3.3 
Interventions 
5 
3.4 
Methodology 
5 
3.5 
Primary outcomes 
5 
3.6 
Secondary outcomes 
5 
3.7 
Hypothesis 
6 
4 
Introduction 
6 
5 
Study objectives and endpoints 
11 
5.1 
Study objectives 
11 
5.2 
Endpoints 
12 
6 
Study methods 
12 
6.1 
General study design and plan 
12 
6.2 
Study setting 
12 
6.3 
Study population 
14 
6.4 
Inclusion and exclusion criteria 
15 
6.5 
Interventions 
15 
6.6 
Randomization 
19 
6.7 
Blinding 
19 
6.8 
Consent 
20 
6.9 
Study Team 
20 
6.10 
Clinic Flow 
21 
6.11 
Defaulter Tracing 
23 
6.12 
Study Assessments 
23 
6.13 
Subgroup enrollment 
24 
6.14 
Intervention adherence and side effect assessments 
25 
7 
Ethical considerations 
25 
8 
Sample Size 
26 
9 
Protocol deviations 
26 
9.1 
Food group stock-outs 
27 
9.2 
Missing Problem Solving Assessment videos 
30 
10 
Study pause 
30 
11 
Procedures for neurocognitive testing 
30 

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11.1 
Modified Willatts problem solving assessment 
31 
11.2 
Malawi Developmental Assessment Tool 
32 
11.3 
Saccade eye-tracking assessments 
33 
12 
Biochemical fatty acid profile analysis 
34 
13 
General analysis considerations 
37 
13.1 
Data cleaning 
37 
13.2 
Outliers 
37 
13.3 
Framework 
37 
13.4 
Timing of analyses 
37 
13.5 
Confidence intervals and p-values 
37 
13.6 
Analysis Populations 
37 
13.7 
Covariates and Subgroups 
38 
13.8 
Missing Data 
38 
13.9 
Multiple Testing 
38 
14 
Subject disposition 
39 
Enrollment 
39 
Allocation 
39 
Follow-Up 
39 
Analysis 
39 
15 
Demographic and derived variables 
39 
15.1 
Baseline variables 
40 
15.2 
Derived variables 
41 
16 
Outcome analysis 
41 
16.1 
List of outcomes with variable type 
41 
16.2 
Outcome variable definitions 
42 
16.3 
Primary outcome analysis 
43 
16.4 
Secondary outcome analysis 
43 
17 
Safety Analyses 
44 
18 
Quality assurance of statistical programming 
44 
19 
Attachments 
44 
20 
References 
53 
 
 
 
 
 
 
 
 

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2 Abbreviations and Definitions 
AE 
Adverse Event 
ALA 
Alpha-linolenic acid 
ARA 
Arachidonic acid 
DHA 
Docosahexaenoic acid 
DHA-HO-RUTF 
High oleic acid RUTF with added DHA 
EPA 
Eicosapentaenoic acid 
HAZ 
Height-for-age z-score 
HO-RUTF 
High oleic acid RUTF 
LA 
Linoleic acid 
MAM 
Moderate acute malnutrition 
MDAT 
Malawi Development Assessment Tool 
MUAC 
Mid-upper arm circumference 
PUFA 
Polyunsaturated fatty acid 
RUTF 
Ready-to-use therapeutic food 
SAM 
Severe acute malnutrition 
SRT 
Saccadic reaction time 
S-RUTF 
Standard ready-to-use therapeutic food 
WAZ 
Weight-for-age z-score 
WHZ 
Weight-for-height z-score 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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3 Trial Summary 
3.1 Type of study 
Prospective, randomized controlled blinded clinical trial 
3.2 Problem to be studied 
Cognition in children with severe acute malnutrition (SAM) treated with ready-to-use 
therapeutic food (RUTF).  
3.3 Interventions 
Three different RUTF formulations will be compared: standard RUTF (S-RUTF), high oleic 
RUTF (HO-RUTF) made with high oleic peanuts and vegetable oils, HO-RUTF with added 
docosahexaenoic acid (DHA-HO-RUTF). 
3.4 Methodology 
2758 children in Southern Malawi aged 6-59 months with uncomplicated SAM were 
randomized to receive S-RUTF, HO-RUTF, or DHA-HO-RUTF that provides 175 
kcal/kg/day for up to 6 bi-weekly follow-up visits (or 12 weeks absent defaults). 
Anthropometry, demographics, symptom assessment, and past health information were 
recorded at enrollment. Anthropometry, compliance, and symptom assessments were 
repeated fortnightly until an outcome was reached. A plasma sample was obtained from a 
subset of children after 4 weeks of treatment. Recovery was defined based on the 
anthropometric criteria that were used to diagnose SAM. Default was defined by missing 3 
consecutive visits. Death was recorded when reported by the caregiver. Children who did not 
recover after 6 follow-up visits were designated as “remained malnourished,” specified as to 
whether they had reached criteria for moderate acute malnutrition (MAM), or remained with 
SAM. 
 
Neurocognition was assessed in a subset of participants using three tests: 
• Modified Willatts problem solving assessments: intentionality was assessed in 
constructed scenes in which a child could retrieve an object with and without barriers 
between them and the object. This was assessed within 4 weeks of completion of 
nutritional therapy. 
• Malawi Developmental Assessment Tool (MDAT): a standardized 4-part exercise, 
assessing gross motor, fine motor, language, and social skills. MDAT was assessed 6 
months after completion of nutritional therapy. 
• Saccadic eye-tracking assessment: an electronic, laptop-based tool that administers a 
visual paired comparison memory task and infant orienting with attention saccade 
task. This was assessed 6 months after completion of nutritional therapy. 
3.5 Primary outcomes 
MDAT global z-scores and modified Willatts problem solving assessment intention scores  
3.6 Secondary outcomes 
Saccadic eye-tracking assessments, recovery, fatty acid percentages in plasma, MDAT sub-
domain z-scores, rates of gain in weight, mid-upper arm circumference (MUAC), and length, 

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death, default, adverse reactions, adverse events 
3.7 Hypothesis 
Children with SAM treated with HO-RUTF or DHA-HO-RUTF will have superior cognitive 
performance than those treated with S-RUTF. 
 
 
4 Introduction 
4.1 General Background 
Undernutrition is a global problem implicated in nearly 50% of the approximately 5.6 million 
deaths of children under 5 years worldwide each year.1 Despite the success of humanitarian 
interventions, over 200 million children under 5 years continue to suffer from stunting and 
wasting as a consequence of undernutrition.1,2 Of the children who survive, lifelong 
morbidities including neurodevelopmental insults may persist despite the apparent physical 
recovery.2,3 Long-term follow-up of those who recover from undernutrition has revealed 
lower educational achievement, lower cognitive scores, and intellectual and learning 
disabilities throughout adolescence and into adulthood.4-6 Sustained cognitive and 
psychological deficits stemming from early life malnutrition contribute to lower human 
capital and adult productivity, which may further aggravate social and economic challenges 
for individuals in disadvantaged communities, contributing to a cycle of intergenerational 
poverty.4,5,7 These findings highlight the importance of understanding the neurocognitive 
changes associated with undernutrition and developing interventions that optimize brain 
development along with promoting physical growth. 
 
Currently, community-based treatment for uncomplicated severe acute malnutrition (SAM) is 
the prescribed feeding of ready-to-use therapeutic food (RUTF). RUTF is a micronutrient-
fortified, lipid-rich (45-60% of total energy), high-energy food (520-550 kcal/100 g) 
formulated to support rapid growth and promote weight gain in affected children. 
Formulations of RUTF commonly consist of four main ingredients: peanut butter, milk 
powder, vegetable oil, and sugar, with added vitamins and minerals. Although there are 
standards set by the World Health Organization, RUTF ingredients will vary depending on 
local availability and cost. Currently, the WHO standards stipulate that RUTF’s nutritional 
composition is to match the F-100 therapeutic milk, a “catch up growth” formula, used to 
treat SAM in hospitals.3 While macronutrient profiles are well described, specific fatty acid 
requirements of RUTF formulations have not been standardized. As a result, the use of 
locally available oils and conventional peanuts often result in RUTF formulations with fatty 
acid profiles that do not support optimal brain development.   
 
4.2 RUTF fatty acid composition suppresses omega-3 synthesis 
When RUTF was originally formulated, energy density was maximized by the inclusion of 
large amounts of lipid. This strategy also created a product with low water activity to avoid 
bacterial proliferation. Due to their wide availability and high-fat content (78% of total 
energy from fat), peanuts were chosen as the base of RUTF. At the time of its development, 
the only fatty acid composition specification given for RUTF was that the omega-6 
polyunsaturated fatty acid (PUFA) should make up 3-10% of total energy and omega-3 
PUFA make up 0.3-2.5% of total energy. The United Nations Children’s Funds (UNICEF) 
has additionally specified that the omega-6 to omega-3 PUFA ratio to be less than 10:1, as a 

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dietary recommendation made for all healthy humans. 
 
Most RUTF formulations are made with peanuts and additional readily available processed 
oils. Conventional peanuts, the base of RUTF, are high in the omega-6 PUFA linoleic acid 
(LA, 18:2n-6; 25-40% of total fatty acids) and contain negligible amounts of the inefficient 
precursor of active omega-3 PUFA, alpha-linolenic acid (ALA, 18:3n-3; 0.01-0.4% of total 
fatty acids).8,9 Additional oils from sources such as soy, sunflower, corn, cottonseed, and 
safflowers are common ingredients in processed foods, including RUTF. Most common oils 
are similarly high in LA and have low or negligible ALA, 9-11 whereas palm oil is 
predominantly comprised of saturated and monounsaturated fatty acids.9 As a source of ALA, 
canola oil (3-10% of total fatty acids) may be added to RUTF to improve the fatty acid 
profile, but even in canola oil the greater amount of LA compared to ALA further increases 
LA.11 Our calculations of oil composition of conventional RUTF formulations find omega-6 
to omega-3 ratio to be always greater than 10:1, and as high as 53:1. Notably missing from 
the vegetable oils in RUTF are long-chain PUFA. As a result, children treated for SAM with 
RUTF rely completely on the endogenous synthesis and secretion to maintain circulating 
levels of conditionally essential long-chain PUFA; however, this very mechanism is inhibited 
by the high omega-6 LA content of standard RUTF. 
 
The long-chain omega-6 PUFA arachidonic acid (ARA, 20:4n-6) and omega-3 PUFA 
docosahexaenoic acid (DHA, 22:6n-3) are synthesized by a series of elongation and 
desaturation reactions from shorter chain precursors LA and ALA, respectively. These 18-
carbon precursor fatty acids compete for the same enzymes catalyzing the elongation and 
desaturation steps, and as a result the overabundance of omega-6 competitively inhibits the 
synthesis of the omega-3.12 As evidenced from experimental animal studies, excessive dietary 
intake of LA compared to ALA overwhelms the pathway and suppresses circulating levels of 
DHA.13 The same is true for conventional formulations of RUTF.14-16 
 
In two separate blinded, randomized clinical trials measuring circulating DHA levels in 
children with SAM being treated with various RUTFs, those on conventional RUTF 
formulations experienced dramatic decreases in DHA from baseline measures.14,16 In a study 
of 61 children with SAM, 21 randomized to receive a standard RUTF with a LA: ALA ratio 
of 11.2:1, Jones et al. reported a significant 25% decrease in red blood cell DHA levels after 
4 weeks of treatment with the standard RUTF.16 Similarly, Hsieh et al. reported a significant 
25% decrease in plasma phospholipid DHA levels after 4 weeks of treatment with a control 
RUTF (LA: ALA ratio of 53:1).14 However, with a specially formulated, balanced dietary 
provision of LA and ALA (LA: ALA ratio = 1:1) using high-oleic peanuts, Hsieh et al. found 
plasma phospholipid DHA concentrations after 4 weeks of treatment remained unchanged 
from baseline. Thus children’s endogenous synthesis of DHA was sufficient to maintain an 
adequate amount of DHA when not challenged with overwhelming omega-6 LA.14 The 
substantial decreases in circulating DHA levels in children treated with standard RUTF 
formulations are of serious concern, as DHA supply to the brain is critical to support optimal 
neurocognitive development. 
   
4.3 Brain development and docosahexaenoic acid 
Brain development is vulnerable to environmental insults, such as dietary deficiency and 
more specifically insufficient omega-3 fatty acid intakes.6,17 Surprising to many, the brain is 
one of the fattiest organs in the human body with a lipid content exceeding 50% by dry 

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weight.18 As is the case with virtually all tissues, the fatty acid environment of the brain is 
highly dynamic and supports much more than merely providing the structural components of 
cell and organelle membranes.  
 
Docosahexaenoic acid comprises over 40% of total brain PUFA and has been shown to 
participate in the regulation of cell-survival and signal transduction, mediate 
neuroinflammation and neurogenesis and regulate blood-brain barrier permeability.19,20-22 
During the third trimester of gestation, the brain reaches its maximal accretion rate for DHA, 
which continues to be high through approximately age two, a critical period of brain 
development.23 The brain continues to rapidly accrue DHA through childhood before 
plateauing during adolescence and young adulthood.24 A constant and adequate supply of 
circulating DHA is critical during this period as the brain cannot efficiently synthesize DHA 
from shorter chain fatty acid precursors. Although ALA rapidly enters the brain, ≤ 0.2% is 
converted to DHA.25 Furthermore, during this period the brain undergoes substantial 
structural and functional changes. Advanced neuroimaging studies have shown the 
morphological changes and rapid development of functional neurocognitive systems 
occurring during the first 5 years of life.26 The functional networks and connectome patterns 
established early in childhood influence cognitive function and behavioral skills throughout 
life.  
 
Animal models of omega-3 deficiency demonstrate the significant negative functional and 
behavioral consequences of inadequate DHA during brain development. Diets used to 
establish omega-3 deficiency are often formulated with sunflower, safflower, corn, or peanuts 
oils and exhibit similar fatty acid profiles as some standard RUTF formulations.11,14 In the 
absence of dietary DHA, brain DHA levels may be supported by synthesis from ALA; at 
levels ≤0.8% of total fatty acid on a background of 23-24% LA, however, brain DHA levels 
decrease dramatically.27 At these levels of omega-3 deprivation, the brain reacts to preserve 
DHA through the widespread reduction in the activity of key DHA metabolizing enzymes 
(e.g. calcium-independent phospholipase A2 IV, cyclooxygenase 1), significantly reducing 
brain DHA turnover.28 Brain metabolic changes underlie the observed widespread functional 
neurological deficiencies associated with omega-3 deprivation, such as deficits in reflexes, 
maze learning, motor development, aggression, impulse control, and learning, among 
others.11  
 
The importance of DHA in brain development is further supported through functional 
neuroimaging studies. Studies in children show that DHA supplementation has measurable 
effects on brain activity which may translate to improved learning and behavioral skills.29,30 
Lacking from the literature, however, is any study on the neurocognitive support of DHA in a 
population suffering from malnutrition. Given that children with SAM have a high nutrient 
demand during recovery, which coincides with active brain development, they are likely to be 
at high risk of neurocognitive deficits resulting from inadequate omega-3 intake and 
conversely might experience the greatest benefits from supplementation. 
 
4.4 Dietary strategy to increase circulating DHA levels 
Increasing intake of ALA or other shorter chain omega-3 fatty acids such as eicosapentaenoic 
acid contributes to altering blood DHA levels.31 In healthy humans, targeted increases in 
circulating DHA levels can only be achieved through two approaches: providing preformed 
DHA in the diet, or by reducing dietary LA levels.15,32  

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In the last 20 years, plant breeding with a focus on oil composition has produced high oleic 
soy and peanut with lower omega-6 fatty acids.33,34 The use of high oleic variety ingredients 
over commodity alternatives results in reduced LA levels and a more balanced fatty acid 
profile of RUTF. In a study by Hsieh et al., the balance of omega-6 and omega-3 fatty acid 
profile of RUTF was achieved through the use of high oleic peanut and flaxseed oil.14 After 4 
weeks of feeding high oleic-RUTF, plasma DHA levels, and weight-for-height z scores were 
higher than controls receiving a standard formulation. There were no significant differences 
in anthropometry, recovery, and growth rates between groups, demonstrating that high oleic-
RUTF was well tolerated and did not compromise recovery.14  
 
In addition to increasing omega-3 content of RUTF through added flaxseed oil, supplemental 
fish oil appears to be well tolerated by children with SAM.16 The addition to fish oil as a 
source of dietary DHA at 0.42% of total fatty acids on top of a flaxseed-based RUTF resulted 
in a 17% increase over baseline red blood cell DHA levels after 12 weeks and no significant 
differences were measured with respect to safety outcomes.16 
 
Although previous studies have proven that alternative RUTF formulations with high oleic 
peanut and added DHA can successfully target circulating DHA levels, none have evaluated 
the associated impact on neurocognitive development. The current study aims to investigate 
the effect of a novel high oleic-RUTF and high oleic RUTF with added DHA on 
neurocognitive performance in children with uncomplicated SAM living in rural southern 
Malawi. 
 
4.5 Cognitive assessments  
The primary endpoints of this study are cognitive developmental outcomes which are 
challenging to assess in infants, especially in rural and low-resource settings. We will use 
three separate tests to capture different facets of development and cognition, aiming for a 
comprehensive assessment. One test, the Malawi Developmental Assessment Tool, was 
developed in Malawi as a culturally sensitive test of global development and was validated in 
over 1,500 children. A second, a modified Willatts problem solving assessment, has low 
technological demands, focuses on executive function and intentionality, and has been shown 
to respond to long-chain polyunsaturated fatty acid supplementation. The third, an automated 
saccade eye-tracking assessment, uses looking behavior as a window into attention and 
novelty preference, and is technologically advanced but has been successfully used in rural, 
low-resource settings. As detailed above, there is reason to suspect the fatty acid profile of S-
RUTF is not optimal for promoting cognitive development. In this study, we will investigate 
the effects of novel HO-RUTF and DHA-HO-RUTF on neurocognitive performance in 
children following treatment of uncomplicated SAM living in rural areas in southern Malawi. 
Details of the tests are below. 
 
4.5.1 Modified Willatts problem solving assessment 
Problem solving, or “means-end behavior”, is an important component of cognitive 
development in infants. Means-end behavior specifically involves “the deliberate and planful 
execution of a sequence of steps to achieve a goal.”35 The key cognitive element drawn out 
by means-end testing is intentionality, wherein children must complete subgoals to allow the 
possibility of achieving their final goal. Planning and persistence are essential to success. 
Testing this domain in infants often involves the use of barriers placed between them and 

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their ultimate goal, such that they must plan and intentionally act to achieve the goal. 
Examples of barriers used in prior research in this field include removing a cover or pulling a 
cloth to obtain an object resting on it.36,37 
 
The problem solving assessments used in this study are modified from a protocol created by 
Willatts et al. 35 These tests aim to quantify problem solving such that test scores will reflect 
the infant’s level of cognitive sophistication. They do so by presenting a protocolized series 
of problems to the infants wherein different barriers are placed between the child and their 
goal of obtaining a toy. The purpose of the test is to assess intentionality in solving the 
problems, which involve pulling the toy closer by grabbing a cloth, uncovering the toy, or a 
combination of both of these.  
 
This specific problem solving assessment tool was chosen for multiple reasons, including 
prior validation, low technological requirement, ease of training assessors, standardized 
coding procedures, and its use in research wherein the investigators assessed neurocognitive 
development in response to long-chain polyunsaturated fatty acid supplementation.38 In this 
study, 3-month-old infants were provided with one of two infant foods, one of which 
contained long-chain polyunsaturated fatty acids while the other did not. Follow-up intention 
testing at 9 months of age identified a trend toward improved intention scores and number of 
solutions.  
 
4.5.2 Malawi Development Assessment Tool 
Developmental assessment tools produced in Europe and the United States have been 
translated and adapted for use in other settings but are constrained by multiple factors, 
including culture-specific tasks and items, technological requirements, and limited validation. 
Investigation of several of these tools in a rural Malawian setting showed good reliability for 
some aspects of development, such as gross and fine motor skills, but poor reliability for 
social items.39 The Malawi Developmental Assessment Tool has been developed and studied 
over the past decade to address these limitations.  
 
MDAT is composed of four domains that aim to summarize key aspects of development: 
language, social, gross motor, and fine motor. To perform the test, a trained assessor elicits a 
series of actions by the child and asks the caregiver questions about their child. These tasks 
and questions have pictorial representations to aid in understanding. Assessment items were 
generated via qualitative studies including focus groups of villagers and professionals in 
Malawi.40 Pilot studies were done to operationalize and test these items. Thereafter, a large-
scale community study was performed, including over 1500 children, to test the reliability 
and validity of MDAT and to establish normal reference ranges.41 It showed excellent 
reliability across different observers and over time, as well as high sensitivity (97%) and 
specificity (82%) for neuro-disability.  
 
MDAT has been tested in undernourished children in multiple studies. In a field study in 
Malawi, children with marasmus were tested and found to have substantially higher rates of 
failure, with lower scores in all domains except social development.41 Stunting and wasting 
were associated with significant reductions in approximately half of the MDAT tools, 
suggesting MDAT is sensitive to undernutrition. A study of children hospitalized with SAM 
in Malawi identified profound delays across all domains, including significantly worse 
language scores in those with kwashiorkor compared to marasmus.42 A study in Burkina Faso 

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investigated the impact of different supplemental foods in the treatment of moderate acute 
malnutrition (MAM) using the MDAT and found improvements in multiple domains.43 A 
separate study in Burkina Faso investigated correlates with MDAT in MAM and found that 
higher anthropometrics and omega-3 PUFA levels correlated with higher MDAT scores.44 
Taken together, MDAT has been used by a host of different research groups in various 
settings over the past decade and has been shown to be reliable, valid, and sensitive to 
undernutrition.  
 
4.5.3 Saccade eye-tracking assessment 
Infant looking behavior has been studied as a developmental measure for decades across 
many contexts. An infant’s ability to orient their gaze to novelty and hold their gaze on key 
sources of information offers insight into their attention and memory, is important for their 
ability to interact with their environment, and emerges during the first six months of life.45-48 
Visual orienting speed in infancy has been associated with future executive function and 
performance IQ, and reduced orienting to faces has been associated with social-behavioral 
problems at 4 years of age.49-51 Prior research has suggested looking behavior may be more 
sensitive than global developmental measures.52 Our research group recently studied visual 
orienting in the context of moderate acute malnutrition in Sierra Leone and found a 
preliminary association between therapeutic feeding and reduction in saccade reaction time 
(manuscript in press).  
 
The replacement of manual methods of eye-tracking assessment with automated, 
technologically advanced methods has benefited the field. These methods take advantage of 
the fact that the cornea of the human eye reflects infrared light, which can thus be used to 
track eye movement using a camera. In general, these automated tests use a display screen to 
show the child's attention-grabbing images in different locations across the screen. Software 
calibrates the location of the child’s eyes in relation to the monitor and links the display 
monitor to the eye tracker, thereby allowing the software to identify the location of the 
stimulus on the display and whether the infant’s gaze has fixed on that location. While 
initially these techniques were limited to high-resource settings, more recently they have been 
expanded for use in low or middle-income settings.53,54 A recent study in Malawi used a fully 
automated system for eye-tracking to assess the effect of supplemental feeding on 
development in 6-9-month-old infants. We have adopted this system with the help of its 
primary developer for the improved PUFA study.   
5 Study objectives and endpoints 
5.1 Study objectives 
5.1.1 Broad 
To compare cognitive-developmental measures in children treated for SAM with 3 
formulations of RUTF with different fatty acid compositions. 
5.1.2 Specific 
To compare problem solving assessment intention scores, Malawi Developmental 
Assessment Tool (MDAT) scores, saccade eye-tracking assessment scores, 
biochemical plasma fatty acid profiles, recovery, growth, infectious symptoms, 
mortality, and adverse events in 6-59 month-old children with SAM treated with S-
RUTF, HO-RUTF, or DHA-HO-RUTF in a 7 visit / 12-week home-based therapeutic 

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feeding program. 
5.2 Endpoints 
5.2.1 Primary 
• Malawi Developmental Assessment Tool global z-score 
• Modified Willatts problem solving assessment intention scores 
5.2.2 Secondary  
• MDAT sub-domain z-scores: gross motor, fine motor, language, and social  
• Nutritional recovery, defined as resolution of nutritional edema for at least 2 weeks, 
and WHZ > -2 or MUAC ≥ 12.5 cm depending on criteria used to diagnose SAM at 
enrollment 
• Serum levels of fatty acids, including oleic acid, eicosapentaenoic acid (EPA), 
docosahexaenoic acid (DHA), alpha-linolenic acid (ALA), linoleic acid (LA), and 
arachidonic acid (AA) 
• Saccade eye-tracking assessment scores 
• Rates of gain in weight, mid-upper arm circumference (MUAC), and length 
• Mortality 
• Defaults 
• The average number of days of fever, diarrhea, poor intake during treatment 
6 Study methods 
6.1 General study design and plan 
This was a prospective, individually randomized controlled clinical trial. All endpoints were 
compared using a superiority framework. The control type was standard of care, S-RUTF 
with amoxicillin for 7 days. The intervention arms were HO-RUTF with 7 days of 
amoxicillin and DHA-HO-RUTF with 7 days of amoxicillin. The participants, caregivers, 
investigators, and outcomes assessors were blinded. Participants were randomized 
individually before treatment initiation 1:1:1 to the three study arms. Screening, inclusion, 
and active treatment started in October 2017 and ended December 2020. Each participant was 
followed until a programmatic outcome (recovery, death, default, transfer to inpatient 
facility) or 6 bi-weekly follow-up visits if they remained acutely malnourished despite 
treatment, whichever occurred first. If the participant remained malnourished after 6 follow-
up visits, they were referred to their local health center for evaluation. 
6.2 Study setting 
The study was initially planned to take place at 20 rural health clinics: Chamba, Chikonde, 
Chikweo, Chipolonga, Makhwira, Matiya, Mauwa, Mayaka, Mbiza, Milonde, Mitondo, 
Mlomba, Muloza, Namasalima, Naphimba, Ndakwera, Nkalo, Nkhate, Nsanama, and 
Zombasalima. These sites are located in 5 districts: Chikwawa, Chiradzulu, Machinga, 
Mulanje, and Zomba. Most people who live in these areas farm for subsistence, with maize as 
the staple crop and main source of nutrition. Water is collected from boreholes or rivers 
several times per day. Homes are built of mud bricks, often single-room, and roofed with 
thatch or occasionally corrugated metal. Electricity and plumbing are unavailable, and 
defecation occurs outdoors or in pit latrines. With rare exceptions, women perform household 
labor and child-rearing, and most are illiterate.  

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Sites were accessible by vehicle and contained a building with a roof. Sites were chosen in 
consultation with district health officials and village elders and leaders, including chiefs and 
health surveillance assistants. In general, these sites had limited access to other treatment 
programs and were known from prior research experience to contain populations at risk for 
severe acute malnutrition.  
 
Protocol addendum 
In 2018, 5 enrollment sites were added: Kasinthula, Makokola, Milepa, Nasfan, and 
Thumbwe. In 2018, study enrollment stopped at Nasfan. In 2019, 2 enrollment sites were 
added, Dolo and Nkumaniza. In 2019, enrollment was discontinued at Chamba, Kasinthula, 
Mauwa, Nkalo, and Thumbwe. In 2020, Bondo was added as an enrollment site. In total, 
study participants were enrolled from 28 clinic sites: Bondo, Chamba, Chikonde, Chikweo, 
Chipolonga, Dolo, Kasinthula, Makhwira, Makokola, Matiya, Mauwa, Mayaka, Mbiza, 
Milepa, Milonde, Mitondo, Mlomba, Muloza, Namasalima, Naphimba, Nasfan, Ndakwera, 
Nkalo, Nkhate, Nkumaniza, Nsanama, Thumbwe, and Zombasalima (map below).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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6.3 Study population 
Malawian children aged 6-59 months with uncomplicated SAM (defined by the presence of 
bipedal pitting edema, and/or MUAC < 11.5 cm, and/or WHZ < -3, no active fever, rapid 
breathing, or altered mental status, adequate appetite) who live in the vicinity of the study 

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sites in rural Southern Malawi. 
6.4  Inclusion and exclusion criteria 
6.4.1 Inclusion criteria 
• Age 6-59 months, rounded to the nearest month 
• MUAC <11.5, and/or weight-for-height z-score < -3, and/or bilateral pitting edema on 
the dorsum of feet 
• An acceptable appetite, as defined by the ability to consume 30 grams of RUTF 
within 20 minutes 
• Presentation to one of the included feeding clinics during the recruitment period 
• Permanent residents in the vicinity of one of the feeding clinics 
6.4.2 Exclusion criteria 
• Participation in a therapeutic or supplementary feeding program in the past 1 months 
• Participation in any other ongoing study or supplementary feeding program 
• Children with a chronic debilitating medical condition, including cerebral palsy, static 
encephalopathy, or congenital heart disease. 
• Children with peanut allergy 
• Children with vision or hearing problems 
6.5 Interventions 
The interventions were specially prepared RUTFs, made by Project Peanut Butter in 
Blantyre, Malawi.55 In this study, three RUTF formulations were compared: DHA-HO-
RUTF, HO-RUTF, and S-RUTF. The formulations of these foods are shown in Table 1, 
while Tables 2 and 3 detail the macronutrient and micronutrient contents, respectively.  
 
6.5.1 PUFA optimized RUTF development 
The goal was to produce two alternative RUTF formulations to achieve improved 
polyunsaturated fatty acid (PUFA) profiles, one with preformed DHA and one without. A 
linear programming (LP) tool was used to consider local commodities while maintaining the 
desired nutrient profile, with a focus on the omega-3 alpha-linolenic acid (ALA) and omega-6 
linoleic acid (LA) nutrient constraints.56 The LP tool is designed to aid in the development of 
alternative RUTF formulations by pulling upon a database of candidate ingredients, with 
associated nutrient contents. It then calculates potential formulas which meet RUTF nutrient 
requirements of the joint United Nations agency statement in addition to the optimized 
omega-3 and omega-6 constraints.3,57 The UN agency specifications for FA in RUTF are 
omega-6 FA should be 3-10% of total energy and omega-3 FA should be 0.3-2.5% of total 
energy.  
 
The first objective of the formulation process was to generate RUTF with improved omega-6 
and omega-3 content. The candidate ingredient list included milk powders, vegetable oils 
including high-oleic oils, high oleic peanuts, and sugar. The paper formulation process started 
with an adjustment in the constraint for omega-3 FA to target a minimum of 1.5% of total 
energy. To further optimize the formulation, this was followed by dropping the maximum 
percent of omega-6 from energy to meet a favorable threshold for an ideal fatty acid balance 
and acceptable palatability. Potential alternative PUFA optimized RUTF formulations were 
prepared at the bench top level in the food lab at Washington University in St. Louis. Bench 
top production of improved PUFA formulas was evaluated primarily on organoleptic 

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characteristics and production feasibility.  
 
After organoleptic and production feasibility optimization, the optimal high-oleic RUTF 
(HO-RUTF) formulation was selected. Then food-grade DHA and EPA found in MEG-3 
DHA K powder were added to the ingredient list with a target ingredient constraint of 1.5%. 
The MEG-3 is derived from fish oil but is encapsulated to reduce the organoleptic properties. 
Encapsulation is achieved with the seaweed-derived hydrocolloid carrageenan in combination 
with starch. The encapsulation utilizes a double-wall barrier to preserve the omega-3 EPA 
and DHA while preventing tastes or smells to permeate the food matrix. The encapsulation 
also prevents oxidation of the PUFA, rendering a shelf life similar to S-RUTF.  Additional 
refinement of both formulations occurred, ensuring they were as similar as possible except 
for the addition of the DHA powder. 
 
6.5.2 High oleic RUTF (HO-RUTF) 
HO-RUTF was formulated according to sample ingredients and with nominal LC-PUFA 
levels shown in Tables 1 and 2. The formula was chosen from about 20 sample formulas 
developed with our in-house food component/nutrient database and optimization system as 
described above.58 The primary ingredient differences in lipid sources compared to the 
standard RUTF are the replacement of regular peanuts with high oleic (HO) peanuts, the 
addition of a small amount of perilla oil, and the removal of canola oil. Because the objective 
of the formulation process was to lower the LA as much as possible, the formulations were 
pulling less overall lipid from vegetable oils and more from the HO peanut, which also added 
protein. To ensure the formulation remained within the specifications for other 
macronutrients such as protein, a small quantity of the dried nonfat milk was replaced with 
sweet whey powder, which is lower in protein and higher in lactose. The amount of 
emulsifier was decreased to establish optimal suitability for large-scale production. The final 
formulation featured a simple set of readily available ingredients and produced a palatable 
product.59 It achieved the lowest LA practically achievable with common ingredients, was 
well within the UN agency specifications for both LA and ALA, and therefore was a 
completely balanced food raising no concerns over nutritional adequacy.  
 
6.5.3 DHA-High Oleic-RUTF (DHA-HO-RUTF) 
DHA content was formulated based on the natural intake of DHA from breast milk. The 
mean 24 h output of DHA in breast milk was 49 mg over the first year of life in five carefully 
followed mothers.60 Other literature estimated global mean breast milk DHA output to be 110 
mg/d.61 Breast milk eicosapentaenoic acid (EPA) is approximately 25% of the DHA levels 
across nine countries.62 Using these benchmark natural intake levels, we formulated DHA-
HO-RUTF to deliver nominal levels of 100 mg DHA and 25 mg EPA per day. Food grade 
DHA and EPA are readily available from many suppliers, and with care, in formulation and 
storage, the RUTF resisted oxidation.  
 
6.5.4 Standard RUTF (S-RUTF) 
Standard RUTF (S-RUTF) is composed of 4 ingredients in roughly equal amounts; milk 
powder, peanuts, vegetable oils, and sugar. S-RUTF is made from conventional, high omega-
6 PUFA peanuts, as well as palm and canola oils. It meets the UN specifications for omega-3 
and omega-6 PUFA content; omega-3 PUFA constituted 0.3-2.5% of the energy in RUTF 
and omega-6 PUFA constituted 3-10% of the energy in RUTF.63 A multiple micronutrient 
powder was added to this mixture to provide a full complement of vitamins and minerals. 

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These powdered ingredients were mixed into a lipid-rich paste to create a soft, semi-solid 
consistency that can safely and easily be consumed by infants.55 RUTF has very low water 
activity making it difficult to contaminate because it does not support the growth of 
bacteria.64 This low moisture, energy-dense, homogenous lipid matrix, therefore, can safely 
be used at home even if hygiene conditions are not optimal. It is also shelf-stable, requiring 
no refrigeration. 
 
To achieve a homogeneous mixture, a specific mixing procedure was followed.  In large-
scale production, the process illustrated in the figure below summarizes the required steps 
and processing limits.   
Oil and powdered hydrogenated vegetable oil emulsifier, specifically Caravan Trancendim 
180, are melted to about 64°C. The remaining oil and peanut paste are released into the 
ribbon blender and mixed for 6 minutes. The oil/emulsifier mixture is added and mixed for 2 
minutes. The dry ingredients are then dispensed and mixed with the paste ingredients for 10 
min. Heating the oil and the mixing process warms the mixture, thus the temperature in the 
ribbon blender can reach 45°C. A grinding step follows, and depending on the equipment set 
up, the RUTF may be passed through the grinder three times to homogenize and reduce 
particle size. The RUTF is held in a tank until it is pumped to packaging with slow mixing 
using a rotating paddle. The temperature in the holding tank may reach 55°C, and the packing 
temperature is around 43°C.        
 
The World Health Organization, World Food Programme, United Nations System Standing 
Committee, and the United Nations Children’s Fund in 2007 jointly published specifications 
for RUTF to ensure a worldwide supply of effective, safe RUTF products.3 Nutritional and 
microbiological requirements were also subsequently established.63 RUTF must also comply 
with the Recommended International Code of Hygiene Practice for Foods for Infants and 
Children of the Codex Alimentarius, and all mineral salts and vitamins must be listed on the 
Advisory List of Mineral Salts and Vitamin Compounds for Use in Foods for Infants and 
Children, also from Codex.63 All foods were locally manufactured by Project Peanut Butter in 
Blantyre, Malawi, which is fully certified as a RUTF producer by the United Nations 
Children’s Fund (UNICEF). Batches were certified by a battery of 12 microbiological tests 
for which the sampling methodology, test method, and test laboratory are specified by 
UNICEF, primarily testing for Salmonella and Enterobacter spp.65 
  
HO-RUTF underwent acceptability testing before the aforementioned pilot study.14 A double-
blind, randomized assessment of 148 children ages 6-59 months was done wherein half 
received S-RUTF and half HO-RUTF. All children were given 30g of the assigned food and 

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timed during consumption. If after 40 minutes not all food was consumed, the remaining 
amount was weighed, the time recorded, and caregivers completed a survey regarding the 
child’s appetite and likeability of the food. In a second stage, 57 of these children (28 S-
RUTF, 29 HO-RUTF) were provided with 270g of the assigned food, to be consumed in 90g 
increments over 3 days. On the 3rd day, the caregivers completed an additional likeability 
assessment. The results indicated high likeability, with survey results of 5/5 (highest on the 
scale) for 64/74 of those who received S-RUTF and 59/74 of those who received HO-RUTF. 
All 57 caregivers indicated 5/5 scores after the second stage of testing.  
 
The food scientist working for the study team made a variety of prototype recipes for HO-
RUTF and DHA-HO-RUTF from which the final recipes were chosen. These underwent 
informal acceptability testing at the time of bench top preparation, and all were deemed 
equivalent to S-RUTF.  
 
Table 1. Ingredient content of study RUTFs 
Ingredient* 
High Oleic 
RUTF 
DHA High Oleic 
RUTF 
Standard 
RUTF 
Milk powder, non-fat, dry 
22.0 
22.0 
27.3 
Milk, sweet whey powder 
6.1 
6.1 
0.0 
Palm oil 
14.0 
14.0 
16.0 
Canola oil 
0.0 
0.0 
5.1 
Sugar 
26.0 
24.5 
23.0 
High oleic peanuts 
25.0 
25.0 
0.0 
Standard, high omega-6 peanuts 
0.0 
0.0 
23.7 
Perilla oil 
3.0 
3.0 
0.0 
Soy lecithin & a hydrogenated 
vegetable oil emulsifier** 
1.0 
1.0 
2.0 
Micronutrient powder 
2.9 
2.9 
2.9 
DHA powder 
0.0 
1.5 
0.0 
*Content expressed in g/100g RUTF 
**This was a proprietary commercial product, Trancendim 180 (Caravan Foods). 
 
Table 2. Macronutrient content of study RUTFs 
Nutrient* 
High Oleic RUTF 
DHA High Oleic 
RUTF 
Standard RUTF 
Protein, total 
15.2 
15.2 
15.7 
Protein, dairy 
8.9 
8.9 
9.8 
Fat total 
29.5 
30.2 
32.7 
Omega-6 fats 
2.7 
3.1 
6.3 
Omega-3 fats 
1.7 
2.2 
0.5 
DHA, mg 
0.0 
198.0 
0.0 
EPA, mg 
0.0 
40.5 
0.0 
Energy, total 
531.0 
532.0 
541.0 
*Content expressed in g/100g RUTF, except for DHA and EPA which are mg 
 
Table 3. The micronutrient content of RUTF 
Nutrient 
RUTF 

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Vitamin A, mg/100g 
1.07 
Vitamin C, mg/100g  
55.90 
Vitamin D, µg/100g 
28.00 
Vitamin E, mg/100g 
44.50 
Vitamin B6, mg/100g 
2.00 
Vitamin B12, µg/100g 
32.70 
Riboflavin, mg/100g 
3.88 
Niacin, mg/100g 
23.90 
Thiamine, mg/100g 
1.46 
Iron, mg/100g 
14.20 
Zinc, mg/100g 
14.80 
Calcium, mg/100g 
658.00 
Phosphorus. mg/100g 
651.00 
Magnesium, mg/100g 
172.00 
  
6.6 Randomization 
Participants were randomized individually in a 1:1:1 ratio to the three intervention arms. 
Computer-generated block randomization lists were created in blocks of 24, designed using 6 
colors, 2 of which were randomly assigned to each study group using a random number 
generator. One color was inserted each into a small, opaque envelope which was then sealed. 
These opaque envelopes were indistinguishable externally.  
 
At the time of randomization, the caregiver of each participant was asked to randomly select 
one small opaque envelope from the larger manila envelope, thereby assigning the study 
group. Once the group was assigned, this color was written on the top of the participant’s 
study card. 
6.7 Blinding 
Study foods were dispensed in sachets containing 92g of study food and were identical in 
external appearance aside from a colored sticker added to indicate the intervention group. 
Boxes contained 1 of 6 colors corresponding to the randomization group and were labeled 
with that color. As previously described, a pilot study was completed before the initiation of 
the present study to assess acceptability, appearance, and taste of each intervention, with 
attempts made to mask any changes related to HO peanuts and DHA to preserve participant 
and caregiver blinding.14 
 
Team members responsible for test feeding, nutrition counseling, questionnaire completion, 
anthropometric measurements, determination of programmatic outcomes, and neurocognitive 

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assessments were unaware of and did not have access to the allocation code. Team members 
responsible for dispensing the intervention therapeutic foods did so by matching the color on 
each participant’s study card with the box color label. These study members also did not have 
access to the allocation code.  
 
The senior study investigators (Drs. Manary, Maleta, Stephenson) will not have access to the 
allocation code until the study is finished and data analysis has been completed. The 
investigator responsible for statistical analysis (Dr. Stephenson) will take no part in the study 
otherwise, including no clinic visits, measurements, or outcome determinations. Dr. 
Stephenson will be provided with a code that is linked to the study assignment in a separate 
file to which he will not have access. The investigators responsible for calculating the 
problem solving assessment scores, MDAT scores, and saccade eye-tracking assessment 
scores will take no part in the study otherwise.  
6.8 Consent  
Nurses were responsible for describing the purpose and nature of the study to caregivers. 
They would sit with the caregivers away from the center of the clinic and speak 
confidentially. They would describe SAM, RUTF, anthropometric measurements, blood 
draws, and cognitive tests. They would also explain how the study foods were different and 
prior testing that demonstrated acceptability and safety of all study foods. They would 
describe the role of antibiotics in the treatment of SAM. They would assure the caregivers 
that participation was voluntary and that the caregivers could withdraw their children at any 
time for any reason if they so choose, without repercussion and with the assurance of 
continued SAM treatment if indicated. They would inform the caregivers that additional 
children in their household could be treated if malnourished, but not in the research study. 
They described how their child’s data will be kept confidential and protected, with de-
identification when first possible, and assured that any private information they provide 
would be secured and used only for the study in question. They described the tokens of 
appreciation that would be provided to caregivers at study milestones and that these tokens 
were not contingent on continued participation after receipt. They described the potential 
impact of the research and the role caregivers and their children will play in generating new 
knowledge. They described the process of default tracing if the caregiver were not to arrive at 
the clinic, as well as the hope that caregivers would either return to the clinic or send a 
message if their child were hospitalized or had passed away. Finally, they answered any 
questions and then proceeded to explain the mechanics of consent, whether by signature or 
inked thumbprint (consent form attached at end of protocol).  
6.9 Study Team 
Team members included nurses, drivers, research assistants, and neurocognitive assessors. 
Nurses were responsible for assessment for inclusion and exclusion criteria other than 
anthropometric measurements and edema assessment, explanation of study procedures 
including blinding process, appetite test, consenting caretakers, nutritional counseling of 
caregivers at enrollment, and each follow-up with special attention to those who were not 
recovering, identification of children who required transfer to inpatient care, blood draws, 
explanation of follow-up schedule. Drivers were responsible for the distribution of 
intervention foods, distribution of pre-determined tokens of appreciation to caregivers during 
study procedures, and monitoring compliance to study foods. Research assistants were 
responsible for the evaluation of age criteria, measurement of MUAC and length, 

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identification of SAM by WHZ and/or MUAC, and pedal edema assessments. 
Neurocognitive assessors were responsible for problem solving assessment, MDAT, and 
saccade eye-tracking assessments.  
6.10   Clinic Flow  
6.10.1 New child and caregiver arrive at study clinic 
• The child is undressed by the caregiver and weight is measured to the nearest 5 grams 
using a seated digital scale (Seca 334) 
• MUAC measured to nearest 1mm using standard insertion tape 
• Length measured in triplicate to nearest 2mm using rigid length board (Seca 417) 
• WHZ score identified using a WHZ field chart (attached) 
• Pedal edema assessment 
• If the child meets the criteria for SAM, the age eligibility is checked using their health 
passport 
o If the WHZ criterion is met, target weight is calculated using WHZ field chart 
and added to the child’s health passport 
o If they meet the criteria for MAM, they are enrolled in a standard MAM 
feeding program 
o If they are not malnourished, caregivers are told the child’s well-nourished 
status 
• If the child meets age criteria, “PUFA?” is written on their health passport and they 
are directed to the study nurse station 
• A research nurse assesses eligibility based on additional inclusion and exclusion 
criteria 
o If the child appears to have severe acute illness including but not limited to 
rapid breathing, high fever, or altered mental status, they will be transported to 
the closest inpatient nutritional rehabilitation unit 
• The nurse performs test feeding of 30g of RUTF  
o Consumption of the test dose within 20 minutes is required for study 
admission. 
o If the child is unable to consume the 30g, they will be transported to the 
closest inpatient nutritional rehabilitation unit 
• If eligible, the nurse explains study procedures and asks the caregiver if they will 
consent 
o If not eligible but deemed to be safe for community-based management of 
SAM, they will be given 2 weeks of RUTF and asked to return in 2 weeks for 
non-research SAM follow-up 
• The caregiver can either sign their name or place an inked thumb print on the consent 
sheet if they have chosen to participate 
o If they have chosen not to participate, the child will be provided with a 2-week 
provision of RUTF and asked to return in 2 weeks for non-research SAM 
follow-up 
• A nurse explains the randomization procedure and holds out a larger manila envelope 
that contains smaller, individually packaged opaque envelopes that each contain one 
of six study colors, two of which correspond to each intervention 
• Caregiver selects one card at random and opens it to reveal their allocation 
• Intervention allocation is added to a study card, which is then filled out with 

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demographic, anthropometric, health, and illness symptom information 
• The date of the follow-up visit is filled into the card and explained to the caregiver 
• A nurse explains the role of amoxicillin and teaches the caregiver how to administer it 
for seven days 
• The caregiver is directed to study driver station, where the driver determines the 
amount of intervention study food to provide based on a chart with pre-calculated 
sachets to provide 180 kcal/kg/day 
 
6.10.2 Returning caregiver and child arrive at the clinic with a small identification 
study card 
• A full study card is identified in the site binder  
• Weight, MUAC, and length measurements are performed 
• Edema assessment done 
• Research assistant assesses for the fulfillment of recovery criteria 
o If enrolled based on edema alone, resolution of edema for at least 2 visits 
o If enrolled based on WHZ, target weight achievement 
o If enrolled by MUAC, MUAC ≥ 12.5cm 
o If enrolled by both WHZ and MUAC, whichever criterion is met first 
o If enrolled by edema and anthropometric criteria, resolution of edema for at 
least 2 visits and achievement of threshold for recovery for anthropometry 
• If the child has recovered: 
o They are directed to the nursing station for symptom assessment  
o If they are not in the subset to undergo neurocognitive testing, they are 
directed to the driver’s station to receive a token of appreciation for their 
participation 
• If the child has not reached criteria for recovery and they have not yet had 7 visits: 
o They are directed to the nursing station for counseling 
o If the child continues to appear safe to continue community-based treatment, 
symptom assessment performed and a follow-up date given 
o Caregiver directed to driver’s station 
o Intervention food distributed 
• If the child has not reached criteria for recovery and they have had 7 visits: 
o They are given the outcome of “remained malnourished” 
o They are counseled about the next steps and offered a ride to the nearest 
inpatient nutritional rehabilitation unit if the mother consents 
• If the child has reached an outcome as above and is part of the subset to undergo 
MDAT and eye-tracking neurocognitive testing 
o The caregiver is given a date to return for MDAT and saccade eye-tracking 
assessments 
• If the child has reached an outcomes as above and is part of the subset to undergo 
PSA neurocognitive testing 
o They are then directed to the neurocognitive assessor station for problem 
solving assessment or, if unable to take part that day, are given a date to return 
within 4 weeks of SAM treatment outcome date 
o Following neurocognitive assessments, they are directed to the driver station 
for the provision of tokens of appreciation 
 

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6.10.3 Returning caregiver arrives to inform staff their child has passed away or has 
been hospitalized 
• The nurse speaks with the caregiver about the time and cause, if known, of the child’s 
death or reason for hospitalization 
6.11  Defaulter Tracing 
Children enrolled in the research study who were not brought back for their scheduled 
follow-up visit were sought at home by the Community Health Workers via a “send-the-
message” card on which the Research Assistant would transcribe the child’s caretaker’s name 
and directions to their domicile.  The Community Health Worker would be instructed to 
locate the child and ascertain the child’s clinical status by MUAC, presence of edema, and 
general clinical assessment. Acutely ill children would be referred to the health center. The 
child’s caretaker would be encouraged to return to the clinic the next time that the research 
team would be present there. 
 
If the child missed a second follow-up visit, a second “send-the-message” card would be sent.  
If the child missed a third follow-up visit, the entire study team would attempt to locate the 
child after clinic with the aid of the project 4x4 vehicle. If the child was still unable to be 
located, they would be classified as a defaulter for the study. 
 
Caregivers were assured that they could withdraw their child from the study at any point and 
for any reason if they so choose. Study staff discussed the decision with the caregiver if the 
caregiver had concerns. The mother was assured her child would continue to receive 
treatment for SAM if indicated regardless of their decision to withdraw. 
 
Community Health Workers received a small monetary gift and/or cell phone units for any 
successful tracing of children or information obtained via “send-the-message” cards. 
6.12  Study Assessments  
Below is a table detailing the timing of study assessments and procedures if an individual 
were to remain in treatment for the full 7 visits. If alternatively, a participant reached a 
programmatic outcome before visit 7, that outcome week would be the last week of 
assessment and they would not receive therapeutic food at that visit.  
 
Table 4. Study assessments 
Visit 
1 
2 
3 
4 
5 
6 
7 
Week* 
0 
2 
4 
6 
8 
10 
12 
Test feeding** 
X 
 
 
 
 
 
 
Weight 
X 
X 
X 
X 
X 
X 
X 
Target weight 
calculation 
X 
 
 
 
 
 
 
Length 
X 
X 
X 
X 
X 
X 
X 
MUAC 
X 
X 
X 
X 
X 
X 
X 
Edema assessment 
X 
X 
X 
X 
X 
X 
X 
Symptom 
questionnaire 
X 
X 
X 
X 
X 
X 
X 
Socioeconomic 
X 
 
 
 
 
 
 

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questionnaire 
Appetite assessment 
X 
X 
X 
X 
X 
X 
X 
Gross motor 
milestone 
assessment 
X 
X 
X 
X 
X 
 
 
Therapeutic food 
provision 
X 
X 
X 
X 
X 
X 
 
Amoxicillin 7-day 
prescription 
X 
 
 
 
 
 
 
Blood test 
 
 
X 
 
 
 
 
*Approximate, as default visits can extend the number of weeks in the study 
**Can be done any week at the discretion of study nurses if there are concerns about the 
child’s ability to continue community-based treatment 
 
Below is a table of cognitive-developmental assessment timing. These assessments were 
performed on a subset of study participants as determined by sample size requirements.  
 
Table 5. Neurocognitive assessment timing 
Visit 
Study outcome* 
6 months post-
outcome** 
Problem solving  
assessment 
X 
 
Malawi Developmental 
Assessment Tool 
 
X 
Saccade eye-tracking 
assessment 
 
X 
*Could fall within 4 weeks of outcome 
**Could fall between 5-7 months post SAM program outcome 
6.13 Subgroup enrollment 
6.13.1 Neurocognitive testing 
Subsets of the study population underwent modified Willatts problem solving assessment 
testing, MDAT, and saccade eye-tracking assessments, as the required samples sizes for each 
were smaller than that of the entire study. For the problem solving assessment, beginning in 
February 2018, all participants under two years of age were assessed with a PSA within four 
weeks of their clinical outcome. Beginning in March 2018, all participants under 30 mo of 
age were asked to return to clinic five to seven months later for testing with the Malawi 
Developmental Assessment Tool and eye-tracking testing. This method allowed for adequate 
power of the neurocognitive outcomes, while not unduly subjecting children to the testing 
procedures.  
 
6.13.2 Plasma sampling 
A subset of the study population underwent a blood draw for plasma fatty acid testing. This 
subset was determined by participation during two designated periods of the study, from 
specified dates in January 2018 to October 2018 and from December 2019 to August 2020. 
During these intervals, all caretakers were asked at all study sites for permission to draw a 
blood sample. It was expected that many caretakers would opt out of the blood draw. The 

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unusual choice for this subset was guided by a local fear in Malawi at the time that health 
workers who were drawing blood were using the blood to spiritually curse the child. This fear 
was so strong that in a context outside of our study, two health workers were murdered for 
blood collection.  
6.14 Intervention adherence and side effect assessments 
6.14.1 Adherence 
When the child was brought back for their first follow-up visit after enrollment in the study, 
the nurse would assess adherence to the intervention from the caretakers. All assessments 
were conducted in a non-judgmental way and it was made clear to the caretakers that we were 
only asking about adherence for the sake of data collection, and that the child’s continued 
care and feeding and the caretaker’s prizes for participation in the study would not be 
affected by the answers to the adherence questions. Assessment of adherence was done using 
2 criteria: 1) verbal report from the caretaker; 2) examination of empty RUTF sachets.  Based 
on these criteria, the nurse would document the number of days of intervention the child 
received on the child’s study data card. 
 
6.14.2 Side Effects 
The nurses also asked the caretakers about any potential associated side effects, including 
(but not limited to) abdominal pain, emesis, rash, and diarrhea. Specific questions about the 
timing of these side effects relative to entry into the study and the duration and severity 
would be asked. If there was any concern that a child may have had a side effect, an adverse 
drug reaction would be reported and discussed with the research team.   
7 Ethical considerations 
In accordance with the Declaration of Helsinki, we aimed to complete the study using the 
highest ethical standards for clinical trials research.66 Ethical approval was obtained from the 
University of Malawi College of Medicine Research and Ethics Committee and the Human 
Research Protection Office at Washington University in St. Louis. Approval was obtained 
from the University of Texas at Austin and Cornell University. Letters of support were 
obtained from the district health officers in each district where the study would be conducted. 
Local village chiefs surrounding the study sites gave their consent and support for the studies. 
Each child’s caregiver was briefed on the study procedures and interventions. Verbal and 
written informed consent was obtained from all caregivers. Caregivers who were unable to 
sign or write their names were asked to document consent using their thumbprints.  
 
On the day of enrollment, caretakers were informed about the process of randomization, the 
duration and requirements of participation in the study, and the benefits of participation in the 
study through an interactive oral explanation. Only caretakers who expressed continued 
interest in the study after being fully informed were enrolled; otherwise, they were enrolled in 
the standard therapeutic feeding programs administered by Project Peanut Butter. 
 
Children who were deemed ineligible to participate in the research study were treated in a 
standard SAM therapeutic feeding program unless they were too ill for community 
management, in which case they were transported to the closest inpatient nutritional 
rehabilitation unit.  
 

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Discussions with district health officers and health service assistants at each site occurred 
before the initiation of the study to address any ethical concerns they had. Individual written 
or thumb print consent was obtained at the time of enrollment.   
8 Sample Size 
The primary outcomes in this study are MDAT and problem solving assessment results, while 
the key secondary outcome is recovery. To determine the sample size needed for this study, 
we first assessed what sizes would be needed for each primary outcome as well as the key 
secondary outcome.  
 
MDAT z-scores were expected to have a standard deviation of approximately 1.1 in this 
population, based on prior research.67-69 An effect size of 0.25 SD would represent a 
clinically meaningful result and using this difference with 25% attrition, parameters of 80% 
power, and two-sided 95% significance for superiority testing, 400 participants were required 
per group. 
 
The problem solving assessment is a modified version of the Willatts test.36 In research 
testing the effect of DHA on infant cognition, Willatts et al. obtained a standardized effect 
size of 0.51 ((7.7 – 6) / 3.35). We did not anticipate an effect size this large in our trial for 
several reasons, including study environment (less well-controlled setting), participants will 
have either just recovered from or will continue to have malnutrition, the supplementation 
period is shorter, and the age distribution will be wider, likely increasing variance. 
Additionally, we could not predict with confidence the distribution of the PSA intention 
scores in our population because of the wider age range and presence of, or recent treatment 
for, SAM. Thus, this sample size calculation was uncertain. We estimated 20% will be unable 
to complete testing despite repeated attempts for a variety of reasons (including acute effects 
of SAM). Using parametric assumptions, and assuming an effect size one-half as large as that 
found in prior research and using parameters of 80% power and two-sided 95% significance 
for superiority testing, 300 participants would be required per group.  
 
Based on prior experience, recovery rates from SAM in this setting are expected to be 
approximately 89%. If present, we aimed to detect superiority at 4%. Using parameters of 
80% power and two-sided 95% significance for superiority testing, 800 participants per group 
were required to detect superiority at 4%. To account for defaults and other data collection 
issues, 900 participants per group were sought for recruitment. Because this secondary 
outcome was deemed essential, and because it required the largest sample size, this 
determined the total study sample size. The cognitive and developmental outcome sample 
size calculations above determined the number of participants included for each test.  
 
Protocol addendum: The protocol initially aimed to enroll 3800 participants, as indicated in 
the initial clinicaltrials.gov listing. This large sample size was chosen to detect a difference in 
anthropometric recovery of 3 percentage points with 95% sensitivity and 80% power. The 
sample size was reduced to 2700 children in March 2019, on the basis that available 
resources would not allow for the achievement of the original sample size.  
9 Protocol deviations 

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9.1 Food group stock-outs 
Due to supply chain interruptions, between March 9-31 2020 both HO-RUTF and DHA-HO-
RUTF were unavailable and participants were only randomized in 1 food group. No high 
oleic peanuts or DHA could be imported. The heavy usage of S-RUTF in March 2020 then 
caused a stock out of S-RUTF from April 8-May 8. During this period, participants were 
randomized to both HO-RUTF and DHA-HO-RUTF. The last stock out period was in 
December 2020, in the final month of the study, which resulted in only HO-RUTF being 
available. All participants enrolled during this time received HO-RUTF. For the first and last 
stock out periods, there was only one food option and all participants were therefore given 
that sole available food. For the stock period in April-May, the remote study member sent a 
new randomization block with only 4 colors (2 foods). Once all 3 foods were in stock, the 
randomization scheme went back to blocks of 24 with 6 colors.  
 
These stock-outs also affected 38 children who had already been randomized to their 
intervention group. These children were excluded from analysis for this reason and numbers 
are provided in the Consort flow diagram in the manuscript.  
 
The March 2020 stock-out occurred when the Malawi Bureau of Standards seized the study 
DHA stock due to exceeded expiration date 1 month prior. Following this, we obtained DHA 
oil from a donation from Wiley.  On the following two pages the technical data sheet for this 
fish oil is presented.  We used it as a direct replacement for the DHA in DHA-HO-RUTF.   
 
The material was received packed in a metal barrel under argon as an air freight shipment, 
these precautions maintained its stability.  When produced into RUTF in May-June 2020, 
ample amounts were made and promptly packaged to avoid problems with degradation of the 
omega-3 PUFAs from storing the opened barrel. 
 

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9.2 Missing Problem Solving Assessment videos 
On 12 August 2019 study staff discovered that an external hard drive that contained video 
recordings of study participants was missing from its storage space at our study office. The 
drive contained video recordings of infants participating in the problem solving assessment in 
the company of their mother or a care giver. The video files in the hard drive are linked to 
study site but not to participant ID. The staff in Malawi tore apart the office to search for the 
missing hard drive with no success in locating the hard drive. A total of 453 study 
participants videos files were completely lost as the files were too large to store on the field 
computers and they had not been backup on any other devices since March 2019. The PSA 
files lost were collected between end of March 2019 and 9 August 2019.  
 
This incident was reported to all necessary institutional review boards and it was determined 
that the risk to the participant was minimal as the participant information that was contained 
in the hard drive is not linked to participant ID although the participant and caretaker faces 
are identifiable. The information in these videos, however, is non-sensitive in nature and 
deemed unlikely to cause significant social harm to the participants and their caretakers.  
 
Following this incident, a series of corrective actions and protocols were put in place to avoid 
further data loss and ensure protection of study participants. These procedures included, 
password encrypting the external hard drives, keeping external hard drives stored in a digital 
code-restricted safe with limited access and video files would be uploaded to the College of 
Medicine Public Health Nutrition Group (COM PHNG) in Malawi server and uploaded to the 
Washington University cloud-based server on a weekly basis. 
 
Documentation of this is available upon request. 
10 Study pause 
On 23 September 2019, we received notice from the institutional review board in Malawi, the 
College of Medicine Research Ethics Committee (COMREC), that the study’s approval had 
lapsed since April 2019 and that due to a misunderstanding of the College of Medicine field 
manager approval for study continuation had not been sought. As a result of this lapse in in 
approval and other citations during an audit conducted in August 2019, the study was 
suspended from enrolling participants until all items were addressed and study suspension 
lifted. On 25 November 2019, COMREC lifted suspension and approval was given to resume 
study enrollment.  
 
Documentation of this was available upon request. 
11 Procedures for neurocognitive testing 
At the start of the study and periodically throughout, all neurocognitive assessors underwent 
training in the administration of the modified Willatts problem solving assessment, Malawi 
Developmental Assessment Tool, and the saccade eye-tracking evaluation by a qualified 
trainer.  

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11.1 Modified Willatts problem solving assessment 
After arriving at the clinic, the neurocognitive assessor would set up the problem solving 
assessment administration station in a room across from the clinic site that would allow for a 
sufficiently quiet environment for testing. The station included a table, two chairs, a supply 
box with toys, cloth covers, and a video camera to capture the entire room. As caregivers 
arrived, eligibility would be confirmed by comparison with a master list and the date would 
be cross-checked to confirm the child was meant to be tested that day. The assessor would 
discuss the child’s current health with their caregiver and if any concerns were raised, the 
caregiver would be directed to a study nurse and asked to return to the clinic at the next visit 
for testing.   
 
The assessor would start by establishing rapport with the child, invoking the caregiver’s 
assistance. The goal was to establish a playful and comfortable environment for the child. 
The assessor would explain the testing procedure to the caregiver, including its purpose and 
the steps involved, as well as the need for recording the test. They would then instruct the 
caregiver to place the child on their lap facing forward across the table and to keep the child 
in this position as best they were able.  
 
Next, the assessor would show the child three different toys, playing with them and offering 
them to the child to play, noting which one the child seemed to prefer best. The assessor 
would allow the child to play with the toys for at least 3 minutes to become comfortable, and 
would then provide the child with the toy they seemed to prefer. The assessor would then 
attempt to retrieve the toy from the child, asking them to return it and reaching out for it, and 
continue with playful attempts until they were successful in assuring the child’s cooperation. 
 
Test 1: cloth and toy 
The assessor would present the cloth to the child, moving it around the table and allowing the 
child to play with it until their interest waned. They would then bring up the toy and assure 
the child was attending to it, after which they would place the toy on the cloth and move them 
around the table together. Next, they would slide the cloth with the toy on top toward the 
child, placing it within 5 cm of the child’s edge of the table, and withdraw to their side. The 
assessor would then watch the child’s response. If the child did not reach for the cloth to pull 
it and the toy toward them to grab the toy, the assessor could say general phrases such as “go 
ahead,” “you can do it,” or “it’s ok,” but not specific phrases like “grab the toy.” The child 
was given 30 seconds for the task. If the child performed the problem solving maturely, the 
assessor could move on to problem 2. If they did not, the assessor could repeat problem 1 one 
more time at most before moving on to problem 2 regardless of success.  
 
Test 2: cover and toy 
The assessor would present the cover to the child in the same manner as above. They would 
then bring up the toy and, confirming the child was watching them, would place the toy under 
the cover. They would then gently slide the cover and toy toward the child to within 2 cm of 
the child’s edge of the table, with the toy under the middle of the cover, before withdrawing 
to their side. The child was given 30 seconds for the task. If the child readily removed the 
cover and grabbed the toy, the assessor would then move on to problem 3. If the child did not 
readily react, similar phrases as above could be used for encouragement, without direct 
instruction. The assessor could repeat problem 2 one more time at most. Reasons for 

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repeating could be lack of interaction with cover or toy, knocking both off the table, seeming 
to swipe at both and accidentally uncovering the toy, or if there was a long delay between the 
presentation of cover and toy and the child’s action. Assessors would stop at problem 2 only 
if the child did not succeed on either problem 1 or problem 2. 
 
Test 3: cloth, toy, and cover 
The assessor would then present the cloth and place the toy on top, confirming the child was 
watching them. They would then place the cover on top of the toy, again confirming the child 
has seen, before sliding the cloth/toy/cover over to the child, within 5 cm over the child’s 
edge of the table. If the child solved the problem in the mature manner (readily pulled the 
cloth toward themself, removed the cover and grabbed the toy), the assessment was complete. 
If the child did not readily solve the problem, words of encouragement could be used similar 
as those above, without specific direction or pointing. If the child did not solve the problem in 
a mature manner, the assessor could repeat problem 3 at most one more time.  
 
The tests were video recorded and sent to the co-principal investigator at Cornell University, 
who oversaw training of coders there. Coders were required to complete training and achieve 
90% agreement by kappa statistic on test video intention scores before coding for the trial. 
Using software, Behavioral Observation Research Interactive Software (BORIS), assessors 
coded tests and tabulated intention scores. In a modification of the procedure of Willatts et 
al,70 problems were coded for two primary components, each on a scale of 0-2 and each 
assessing an aspect of intentionality: orientation and execution. Orientation was defined as 
the quality of shift in attention (visual fixation, bodily orientation) from the toy itself to the 
subgoal in question (e.g., cloth to pull closer, or cover to remove), or means by which the 
ultimate goal could be obtained. Orientation was scored on three levels: direct shift, 
slow/indirect/fumbling shift, and no shift. Primarily in older children, it was possible for a 
child to demonstrate a phenomenon known as “covert attention,” in which the child executed 
the sub-goal without clear change in visual fixation or body position, or did so too fast for 
observation. If the execution was smooth/perfect, the child would receive credit for 
orientation. Execution is defined as the manner in which the child performs motor actions on 
the task materials when confronted with the barrier, including how they manipulate subgoals. 
Execution is scored on three different levels: smooth/perfect execution, fumbling execution, 
or no execution.  
 
For problems 1 and 2, intention scores ranged from 0-4. For problem 3, intention scores 
ranged from 0-8.   
11.2 Malawi Developmental Assessment Tool 
After arriving at the clinic, the neurocognitive assessor would set up the MDAT 
administration station, often in a room across from the clinic site that allowed for a 
sufficiently quiet environment for testing. As caregivers arrived, eligibility would be 
confirmed by comparison with a master list and dates would be cross-checked for accuracy. 
The assessor would discuss the child’s current health with their caregiver and if any concerns 
were raised, the caregiver was directed to a study nurse and asked to return to the clinic at the 
next visit for testing. Included as an appendix is a list of all the MDAT assessments.   
 
The assessor would open the tablet and enter their initials, participant ID, date of collection, 
date of birth of the participant, and caregiver details. The questionnaire automatically shifted 

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to the appropriate starting point for the participant’s age and the assessor would proceed item-
by-item. The MDAT contained 136 items in total. For evaluation of gross motor, fine motor, 
and language development items, the assessor would directly observe the child. Social 
development is evaluated by questions asked of the caregiver. Each item was scored as 
“pass,” “fail,” or “NA.” “NA” was used if the child did not engage sufficiently for the 
assessor to make a determination. If a participant scored “fail” on six consecutive items in 
any single domain, the questionnaire would automatically change to the next domain.  
11.3 Saccade eye-tracking assessments  
The setup for the eye-tracking assessment required several steps to be completed before 
participants arrived. The laptop, monitor, external processing unit, and webcam all were 
connected to power, the external processing unit was connected to the eye-tracker and laptop, 
the laptop was connected to the monitor and webcam, and the study USB drive was 
connected to the laptop. The software was password protected. The assessor then mapped the 
display monitor and eye tracker such that they aligned spatially. 
 
Next, the caregiver and child would be brought into the testing station. Their ID, name, visit, 
the caregiver’s name, and assessor ID would be written on the whiteboard and the 
participant’s ID was entered into the computer. The assessor would align the lab window and 
webcam window so they were able to see both the video of the child and the video of the eye-
tracking test simultaneously.  
 
Next, the assessor would explain the process and purpose of testing to the caregiver. The 
caregiver would be asked to adjust their baby carrier so the child faced forward and sit in the 
study chair. The assessor would then hold the whiteboard in front of the camera, click record, 
and read the information aloud, before removing the whiteboard from view. The first video 
would be played to attract the child’s attention, and different videos could be tried until the 
child was seen fixing their attention on the screen. The caregiver was then asked either to 
look away or close their eyes, and the assessor would adjust the screen to be at eye level and 
55-65cm away from the child’s face.  
 
The assessor would then initiate the eye-tracking calibration, which aimed to match where 
images appeared on the monitor and where the eye-tracking software calculated their 
presence. The software first calculates the distance of the child’s eyes from the monitor, 
following which the assessor would adjust the monitor to be as close to 60 cm away as 
possible. The assessor would then click “start calibration,” which presented an image at pre-
specified locations. The assessor compared the known image location with eye fixation and 
adjusted the screen as needed, re-calibrating after each change. There were multiple videos 
available for calibration if the initial options did not work.  
 
Next, the assessor would begin testing by showing an “attention grabber,” meant to focus the 
child’s eyes on the center of the screen. They could shake a rattle behind the screen if the 
child would not attend there but could not use a rattle or any other external stimulus except 
during an “attention grabber” step. The software would then run through testing with 
interspersed attention grabbers. The recording could be stopped if the child was not able to 
attend to the screen.  
 
Testing included visual paired comparison task and infant orienting with attention task. 

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Identical procedures to those previously described were used.69 The visual paired task 
consisted of 4 trials of African faces. The infant orienting task measured saccadic reaction 
time in a standardized manner. The testing sequence for paired comparison task and orienting 
attention task was randomized. 
 
After the testing was complete, the assessor would show a sample video to the caregiver to 
explain what was done. They would release the caregiver and then save the file in a 
prespecified format. The assessor would then complete a form that included information 
about the child’s perceived mood and activity level and contained a free comments section 
where they could record technical issues or anything else unusual that occurred during 
testing.  
 
12 Biochemical fatty acid profile analysis 
To evaluate the influence of study RUTF formulations on circulating fatty acid profiles, gas 
chromatography-flame ionization detection (GC-FID) of fatty acid methyl esters (FAME) 
derived from isolated plasma phospholipids was carried out. 
 
At the 4-week study visit, blood samples for fatty acid analysis were collected into 
ethylenediaminetetraacetic acid treated vacutainer tubes. Plasma was separated from red 
blood cells through centrifugation at 1000-2000 x g for 10 mins, following which it was 
transferred to clean tubes and held at -20°C until samples were received at the Dell Pediatric 
Research Institute. 
The choice of analysis of plasma fatty acids levels over other fractions, such as whole blood 
or red blood cells, was twofold. Firstly, plasma phospholipid DHA levels reach a plateau 
after approximately 4 weeks of supplementation or feeding.31 Second, plasma fatty acids are 
stable for over 1 year when stored at -20°C, whereas whole blood and red blood cell PUFA 
are liable to degradation by iron initiated peroxidation when stored at -20°C.71 
 
From plasma collected at 4-week blood draws, phospholipids were isolated using a three-
phase liquid-liquid extraction similar to the methods described by Vale et al. and adapted in 
house for semiautomated extractions by a Hamilton Microlab STARlet robot (Hamilton 
Robotics, Reno, NV).72 Briefly, an aliquot of plasma is weighed out, to which a mixture of 
hexane: methyl acetate: acetonitrile: water (4:4:3:4, by volume) is added. Samples are mixed 
then incubated at room temperature for one hour. The middle phospholipid containing phase 
is then collected into a new tube, dried under nitrogen, and reconstituted in heptane. 
 
After isolation of plasma phospholipid, samples underwent transmethylation to generate 
FAME for analysis by GC-FID. Transmethylation is required to cleave fatty acids from their 
esterified position on the glycerol backbone of their native phospholipid and results in a 
volatile derivative with good chromatographic properties. For the purposes of this study, 
isolated plasma phospholipids were transmethylated via a one-step reaction adapted from the 
methods of Garcés and Mancha.73 Briefly, a mixture of methanol: 2,2-dimethoxypropane: 
sulfuric acid (85:11:4, by volume) to the solvated phospholipid, followed by a mixture of 
heptane: toluene (63: 37, by volume). Samples are then heated at 80°C for 2 hrs. A saturated 
solution of NaCl is then added, and samples are incubated at 4°C for 3 hrs. The top FAME-
containing layer is then transferred to a clean glass tube, evaporated under nitrogen, and 

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reconstituted in heptane for analysis by GC-FID. 
 
Gas chromatography is a classical analytical technique routinely used for the analysis of fatty 
acids. A complex mixture of FAME is injected onto a capillary column coated with a 
stationary phase. An inert carrier gas serves as a mobile phase as gaseous compounds interact 
with the stationary phase of the capillary column, and specific analyte species elute off of the 
column with a characteristic retention time. With respect to FAME, species are separated 
based on carbon chain length and degree of unsaturation. The area of the corresponding 
chromatographic peaks are related to the abundance of FAME in the sample and are put on a 
quantitative basis using experimentally determined response factors. 
 
For the proposed study, FAME were separated on a 20 m × 0.22 mm i.d. × 0.25 µm film 
thickness BPX70 capillary column (Trajan, Austin, TX) configured in a Shimadzu GC2010 
equipped with a flame ionization detector (Shimadzu Corporation, Kyoto, Japan) 
 
FAME were separated on a BPX70 capillary column (25m × 0.22mm I.D. × 0.25 µm df; 
Trajan, Austin, TX) configured in a Shimadzu GC2010 equipped with a flame ionization 
detector (Shimadzu Corporation, Kyoto, Japan).74 Samples were injected into a splitless inlet 
held at 250°C using a Shimadzu AOC-20i/s autosampler. FAME were separated using a GC 
temperature program as follows, 80°C hold for 2 min, 10°C/min to 174°C hold for 2 min, 
10°C/min to 240°C hold for 10 min, and 5°C/min to 245°C hold for 5 min. The flame 
ionization detector was held at 260°C. Peak area response factors were generated from a 
reference mixture of equal weight FAME (Nu-Chek Prep, Inc., Elysian, MN) and applied to 
peak areas under the curve to yield calibrated FAME profiles.  FAME structural identity is 
positively identified by covalent adduct chemical ionization mass spectrometry75.  Fatty acid 
data will be reported as weight percentages (%wt) of total fatty acids identified. A complete 
profile of 25 fatty acids will be collected for each sample. 
 
The test formulations of RUTF, HO-RUTF, and HO+DHA-RUTF were modified to enhance 
the distribution of omega-6 and omega-3 fatty acids. Moreover, the use of high-oleic peanut 
varieties as the base ingredient contributes to increasing the abundance of oleic acid in study 
RUTF. As such, the plasma phospholipid fatty acids we propose to compare across dietary 
treatments are oleic acid, omega-6, and omega-3 series fatty acids. Specific fatty acids of 
interest are indicated and described below: 
 
12.1 Oleic acid 
Oleic acid is an 18-carbon monounsaturated fatty acid found abundantly in vegetable oils, 
notably olive oil. Within the brain, oleic acid is found predominately in white matter where it 
comprises over 30% of the total fatty acids.18 Oleic acid is a critical component of brain 
myelin which is rapidly accumulated following birth.23  
 
12.2 Omega-6 series 
Linoleic acid an 18-carbon PUFA containing double bonds at carbon nine and twelve from 
the carbonyl end. Humans lack delta-12 desaturase and consequently are unable to 
endogenously synthesize LA, lending to LA being considered an essential fatty acid. Beyond 
acting as a precursor for longer chain omega-6 polyunsaturated fatty acids, such as ARA, LA 
is incorporated into the brain where it plays a role in the neuroinflammatory processes 
through the production of oxidized linoleic acid metabolites (OXLAMs).76 Experimental 

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evidence from rodent studies suggests that diets with high LA contribute to a greater pro-
inflammatory response to brain insult.77 In humans, reducing dietary LA intakes in 
combination with increasing EPA and DHA intake is associated with reduced headache 
duration and severity in patients with chronic daily headaches.76,78 
 
Arachidonic acid is a 20-carbon omega-6 PUFA synthesized from the desaturation and 
elongation of LA, primarily taking place in the liver. During the first two years of life, ARA 
is the most abundant PUFA in the brain.23,79 Over 90% of ARA in the brain is esterified into 
phospholipids; however, free-ARA is released in response to the activation of dopaminergic, 
cholinergic, serotonergic, or N-methyl D- aspartate (NMDA) activation.21 Free-ARA can be 
metabolized by cyclooxygenases, lipoxygenases, and cytochrome P450 to produce bioactive 
eicosanoids which have been shown to mediate neuroinflammatory pathways.21,77,80 
Additionally, ARA is necessary for the synthesis of the two most abundant endocannabinoids 
in the brain, anandamide and 2-arachidnoyl glycerol, which have been shown to regulate 
synaptic function.21  
 
Adrenic acid (AdrA) is the 22-carbon omega-6 PUFA synthesized from elongation of ARA. 
It is present in various tissues including the brain and its concentration increases in the first 
years of life.23 It particularly increases when DHA is low. 
 
Docosapentaenoic acid omega-6 (DPA6) is the 22-carbon omega-6 PUFA synthesized from 
desaturation of AdrA. DPA6 is a structural analog of DHA, with the sole difference being 
that DPA6 has one fewer double bond than DHA. Because DPA6 can be made from dietary 
LA, its concentration increases in DHA deficiency and it has long been known as a marker 
for DHA deficiency. 
 
12.3 Omega-3 series 
Alpha-linolenic acid is the 18-carbon essential omega-3 PUFA. As is the case with LA, 
humans do not possess the required desaturase enzymes for de novo synthesis of ALA; 
therefore, it must be supplied by the diet. As a precursor to longer-chain omega-3 PUFA such 
as eicosapentaenoic acid (EPA, 20:5n-3) and DHA, ALA competes with LA for elongase and 
desaturase enzymes. Excess dietary intakes of LA have been shown to suppress circulating 
DHA levels from ALA by outcompeting ALA for elongase and desaturase enzymes.13 
Although ALA rapidly enters the brain, it does not appreciably contribute to local within-
brain production of DHA.25 Despite sharing a similar incorporation rate as DHA, brain ALA 
levels are maintained very low (≤0.5% of total fatty acids) likely due to rapid β-oxidation.25 
ALA has been proposed as a potential substrate for the production of ketones within the 
brain, which may serve as an important energy source to support rapid brain growth and 
development during infancy and young childhood.81,82 
 
Eicosapentaenoic acid is a product of ALA elongation and desaturation. Despite being 
relatively abundant in the blood, EPA levels are exceedingly low in the brain, about 250-300 
fold lower than DHA levels.83 Similar to ALA, EPA is rapidly β-oxidized after entering the 
brain. Although levels are maintained very low, EPA has been suggested to play a role in the 
regulation of mood.21 Results from randomized clinical trials suggest that supplementation 
with EPA dominant (EPA ≥ 60% of EPA+DHA) omega-3 is effective against primary 
prevention of major depression.84 Additionally, EPA is converted to oxygenated metabolites 
and specialized pro-resolving mediators that have been shown to promote the resolution of 

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inflammation following brain injury.83,85 
 
Docosahexaenoic acid is one of the most abundant PUFA in the brain and has been shown to 
play an important role in the regulation of neuroinflammation, cell survival, synaptogenesis, 
and signal transduction.20,21 Increasing levels of DHA in the blood and tissues can only be 
achieved through either decreasing the intake of LA and maintained adequate intake of ALA, 
or consuming dietary DHA.  
13 General analysis considerations 
13.1  Data cleaning 
All data were double-entered into password-protected Microsoft Access databases and 
differences will be reconciled by reassessing the original data cards and using these as the 
authoritative version.  
13.2  Outliers 
Extreme values of anthropometrics and z-scores will be evaluated individually and assessed 
for obvious recording errors. If no error is found, previously identified criteria will be used to 
exclude these outliers: 20 cm < MUAC < 6.5 cm, weight change > 25g/kg/day, or MUAC 
change > 1.5 cm per week.86 
 
Extreme MDAT z-scores were evaluated individually and assessed for obvious recording 
errors. Outliers that were clearly impossible would be corrected if possible or recoded to 
missing if correction was not possible. Based on observation of the distribution, global 
MDAT z-scores <-5 were excluded.  
13.3  Framework 
All outcomes will be tested using a two-sided superiority framework. 
13.4  Timing of analyses 
We will conduct all analyses blinded to intervention groups by creating a code for actual 
group assignments. The final analysis will be performed after all study participants have 
achieved a programmatic outcome and all data have undergone cleaning. 
13.5  Confidence intervals and p-values 
In all analyses, confidence intervals will be two-sided and calculated at the 95% confidence 
level, and all testing will be two-sided and considered significant at the p < 0.05 level.  
13.6  Analysis Populations 

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13.6.1 Modified Intention-to-treat (ITT) 
This population will include all randomized study subjects with one exception: all 
participants in whom a pre-existing exclusion criterion is found after randomization will be 
excluded from the study and the modified intention-to-treat analysis. This is justified by the 
blinded nature of the experiment and the type of exclusion criteria in question – determining 
age rounded to nearest month < 6 or > 59 mo, presence of a chronic debilitating medical 
condition, vision or hearing problem – which are unlikely to be affected by allocation group 
even in the absence of blinding. All superiority analyses will be performed as modified 
intention-to-treat.  
13.6.2 Protocol deviation subgroup 
We will perform a secondary analysis including only those participants who were enrolled 
outside the windows of the three food group stock-outs that led to unbalanced randomization. 
We will characterize the enrollment characteristics of these children and repeat the primary 
and key secondary analyses to assess for bias introduced by unbalanced randomization. 
 
13.7   Covariates and Subgroups 
Multiple variables are hypothesized to exert an influence on neurocognitive assessments. 
• Child age at the time of assessment 
• Child sex 
• Type of SAM (kwashiorkor, marasmus, marasmic-kwashiorkor) 
• Enrollment HAZ and WAZ 
• Presence of active infection: reported days with fever, diarrhea 
 
The above list will be assessed but is not meant to be exclusionary such that the study 
investigators may generate additional observations and hypotheses during the study. 
13.8   Missing Data 
The percent of participants lost to follow-up between enrollment and outcome assessment 
will be tabulated.  
 
Missing data will be identified using summary statistics and quantified using % of 
participants with the missing variable, and % of measurements, where applicable. Missing 
data will not be imputed. In some cases, if the missing data are essential for inclusion in the 
analysis, e.g., date of birth for age-based z-score calculations, these participants will not be 
included in these analyses.  
13.9   Multiple Testing 
There are two primary endpoints in the study and three intervention arms. To reduce the 
family-wise error rate, only two pairwise comparisons will be made each for the MDAT z-
score and problem solving assessment scores, HO-RUTF vs. S-RUTF and DHA-HO-RUTF 
vs. S-RUTF. These two comparisons have been chosen because of their primary importance 
for the study hypothesis.  
 
Multiple secondary endpoints and analyses will be completed, but we will not plan to 
generate P-values for these comparisons, but rather differences with 95% confidence 
intervals.  
 

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13.10  Reporting conventions 
P-values ≥ 0.001 will be reported to 3 decimal places. P-values less than 0.001 will be 
reported as < 0.001. Percentages will be reported to the tenths digit. Z-scores will be reported 
to the hundreds digit. Estimated parameters, such as regression coefficients, will be reported 
to 3 significant figures. Confidence intervals will be 95%.  
 
14 Subject disposition 
The CONSORT flow diagram (skeleton below) will detail the number of children at 
enrollment, allocation, follow-up, and analysis stages, with details of exclusions between 
each stage listed.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
15 Demographic and derived variables 
Assessed for eligibility (n =  ) 
Excluded  (n =   ) 
• 
Under 6 months of age (n = ) 
• 
Over 59 months of age (n = ) 
• 
Transfer from inpatient nutrition unit (n = ) 
• 
Recently treated as outpatient for acute 
malnutrition (n = ) 
• 
Obvious chronic/debilitating illness (n = ) 
• 
Unable to obtain consent (n = ) 
• 
Not a resident of catchment area (n = ) 
Randomized (n =  ) 
Enrollment 
Analyzed  (n =  ) 
• 
Recovered (n = ) 
• 
Failed (n = ) 
o 
Died (n = ) 
o 
Hospitalized (n = ) 
o 
Remained acutely malnourished (n = ) 
o 
Default (n = ) 
Allocated to HO-RUTF (n =  ) 
Subsequently excluded (n =  ) 
• 
Under 6 months of age (n = ) 
• 
Over 59 months of age (n = ) 
• 
Incomplete data (n = ) 
• 
Actually moderately malnourished (n = ) 
• 
Failed test feeding (n = ) 
• 
Received incorrect food (n = ) 
• 
Transfer from inpatient hospital nutrition unit (n =) 
Allocated to HO-RUTF (n =  ) 
Allocated to HO-RUTF (n =  ) 
Same as central 
branch 
Same as central 
branch 
Allocation 
Follow-Up 
Analysis 

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15.1 Baseline variables 
Category 
Definition 
Demography 
 
Age 
Child age in months; continuous 
Sex 
Male or female; categorical 
Child still being breastfed 
Yes or no; categorical 
Caregiver relation to child 
Mother, father, sibling, grandparent, aunt, 
uncle 
Mother alive 
Yes or no; categorical 
Father alive 
Yes or no; categorical 
Number of siblings of participant 
Discrete 
Number of siblings of participant that have 
died 
Discrete 
Number of radios in the home 
Discrete 
Number of bikes in the home 
Discrete 
Latrine use 
Yes or no, categorical 
Water source 
Borehole, well, or river; categorical 
How many times per day water retrieved 
Discrete 
Animals sleep in the home 
Yes or no; categorical 
Number of individuals that sleep in the 
same room as the child 
Discrete 
Material of household roof construction 
Thatch or metal sheets; categorical 
Health questionnaire 
 
Child with good appetite 
Yes or no; categorical 
Presence of fever in past 2 weeks 
Yes or no; categorical 
Days of fever in the past week if present 
Discrete 
Presence of diarrhea in past 2 weeks 
Yes or no; categorical 
Days of diarrhea in the past week if present Discrete 
Participant had HIV testing done 
Yes or no; categorical 
If above yes, result 
Positive or negative; categorical 
If positive, on antiretroviral therapy 
Yes or no; categorical 
Caregiver had HIV testing done 
Yes or no; categorical 
If above yes, result 
Positive or negative; categorical 
If positive, on antiretroviral therapy 
Yes or no; categorical 
Participant on tuberculosis treatment 
Yes or no; categorical 
Adult in participant’s home on tuberculosis 
treatment 
Yes or no; categorical 
Adherence 
 
Child eating RUTF well 
Yes or no; categorical 
Gross motor development questionnaire 
 
Sitting without support 
Yes or no; categorical 
Hand-and-knees crawling 
Yes or no; categorical 
Standing with assistance 
Yes or no; categorical 
Walking with assistance 
Yes or no; categorical 
Standing alone 
Yes or no; categorical 
Walking alone 
Yes or no; categorical 

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Anthropometry  
 
Weight 
Measured to nearest 5g; continuous 
Length 
Measured to nearest 2mm; continuous 
MUAC 
Measured to nearest 1mm; continuous 
Weight-for-height z-score 
Continuous 
Weight-for-age z-score 
Continuous 
Length-for-age z-score 
Continuous 
Target weight 
Calculated using z-score chart; continuous 
Edema 
Present or absent; categorical 
Edema severity 
1-3+; discrete 
 
15.2 Derived variables 
• Weight gain will be calculated as g/kg/d over the treatment course for all children for 
whom data is available. MUAC gain and length gain will be total mm/wk over the 
treatment course for all children for whom data is available. 
• Because the two intervention arms have overlapping components, i.e. the DHA-HO-
RUTF arm contains DHA in addition to HO (the identifying component of the HO-
RUTF intervention), in a secondary analysis, we will create variables for DHA and 
HO for regression analyses rather than a variable for the intervention group.  
• We will assess the distribution of saccade reaction time results, which have been 
identified as right-skewed in prior analyses. If we identify a right-skewed distribution, 
we will attempt transformations, first log, then square root, and assess for 
improvement in distribution symmetry. 
 
16 Outcome analysis 
16.1 List of outcomes with variable type 
Primary 
 
Problem solving assessment intention 
scores 
Ordinal 
Malawi Development Assessment global z-
score 
Continuous 
Key secondary outcomes 
 
Recovery 
Yes or no; categorical 
MDAT sub-domain z-scores 
Continuous 
Other secondary outcomes 
 
Neurocognitive outcomes 
 
Saccade eye-tracking assessment score 
Continuous 
Anthropometry 
 
Weight change 
g/kg/d, continuous 
Length change 
mm/wk, continuous 
MUAC change 
mm/wk, continuous 
Programmatic Outcomes 
 
Recovery 
Yes or no; categorical 
Death 
Yes or no; categorical 

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Default 
Yes or no; categorical 
Remained malnourished 
Yes or no; categorical 
Transferred to hospital 
Yes or no; categorical 
Biochemical testing, serum levels 
 
Oleic acid 
%; continuous 
Docosahexaenoic acid 
%; continuous 
Eicosapentaenoic acid 
%; continuous 
Alpha-linolenic acid 
%; continuous 
Linoleic acid 
%; continuous 
Arachadonic acid 
%; continuous 
Docosapentaenoic acid 
%; continuous 
Adrenic acid 
%; continuous 
  
16.2 Outcome variable definitions 
16.2.1  Primary outcomes 
• MDAT global z-score for gross motor, fine motor, language, and social domains 5-7 
mo after completion of SAM treatment. 
o We will calculate the raw score on each MDAT sub-domain as the number of 
items the child was observed or reported to be able to perform out of 38 gross 
motor items, 34 fine motor items, 35 language items, and 29 social items. 
o We will then calculate global and sub-domain z-scores using the standard 
published norms.  
o For children in whom one sub-domain is unable to be completed, the 
remaining sub-domains will be used to calculate the global z-score. 
o We will require at least two sub-domains to be completed for a global score to 
be included in analysis 
• Problem solving assessment intention scores 
o These scores are ordinal. The distribution was unknown prior to the study 
because the age range was wide and the children had recently been treated for 
SAM. We considered the possibility of combing the three problem scores into 
a composite score, but the meaning of each problem is different, specifically 
problem 3 is more than a mere combination of problems 1 and 2, and so each 
problem will be analyzed separately.  
o Problems 1 and 2 had scores of 0-4, while problem 3 was scored 0-8.  
o NA was scored when children were unable to engage with the problem 
sufficiently for a score to be obtained, and the reasons for NA outcome were 
documented.  
16.2.2 Secondary outcomes 
• MDAT sub-domain z-scores 
• Recovery: recovery from SAM was defined based on the criteria used for diagnosis: 
o Recovery must have occurred on or before the 6th follow-up visit. 
o In those with nutritional edema only, recovery was the resolution of nutritional 
edema for two consecutive follow-up visits. 
o In those with WHZ < -3, a target weight was calculated on enrollment using 
the child’s length and a WHZ chart separated by sex, such that obtaining this 
weight would result in WHZ ≥ -2. Recovery was determined when the child 

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reached the target weight. 
o In those with MUAC < 11.5 cm, recovery was be determined when MUAC ≥ 
12.5 cm 
o In those with nutritional edema and WHZ/MUAC criteria, recovery was 
defined by resolution of nutritional edema for two consecutive visits and 
reaching either of the WHZ or MUAC criteria above. 
o In those with WHZ < -3 and MUAC < 11.5 cm, recovery was determined 
when the child reached either WHZ or MUAC criteria for recovery 
• Default: determined when a child missed three consecutive visits 
• Remained malnourished: the child did not reach an alternative outcome (recovery, 
transfer to hospital, death, default) and remained malnourished after 6 follow-up visits 
• Death: caregiver reported the child died 
• Transfer to hospital: criteria for uncomplicated SAM no longer reached and field team 
determined child required transfer to inpatient nutritional rehabilitation unit 
• Saccade eye-tracking assessment score: automated output of the program, in units of 
milliseconds 
16.3 Primary outcome analysis 
There are two primary endpoints to be compared in this study: MDAT global z-score and the 
modified Willatts problem solving assessment intention scores. The null hypothesis for 
MDAT z-score will be that the sample means are equal between DHA-HO-RUTF vs. S-
RUTF and HO-RUTF vs. S-RUTF. The null hypothesis for the problem solving assessment 
intention scores will be that the odds ratios for higher scores will be equal between DHA-
HO-RUTF vs. S-RUTF and HO-RUTF vs. S-RUTF. Mean differences and 95% CIs will be 
calculated for pairwise comparisons in MDAT z-score, while ORs with 95% CI will be 
calculated for pairwise comparisons in problem solving assessment intention scores.  
 
16.3.1 Global MDAT z-score   
Student’s t test will be used to compare global MDAT z-scores. Differences in mean 
scores with 95% CIs will be reported alongside P-values.  
 
16.3.2 Problem solving assessment scores 
Age is a known predictor of problem solving assessment results and so will be 
included a priori as a covariate in the regression analysis. We will assess for sparse 
cells and, if present, will combine groups to satisfy this assumption. We will test for 
proportional odds using graphical comparison of the beta coefficients each group 
resulting from binary logistic regressions at cut-points of scores >2. An interaction 
term between age and group will be offered into the model and included if it 
demonstrates a statistically significant effect. Multicollinearity is not expected, as 
only the intervention group and age will be entered as covariates into the regression 
model, and the intervention group is randomly assigned. Odds ratios with 95% CIs 
will be reported alongside P-values.  
16.4   Secondary outcome analysis 
Programmatic outcomes will be summarized as n (%). Anthropometric growth rates will be 
assessed for distribution using histograms; if normally distributed, they will be summarized 
with mean and SD. If the data do not appear normally distributed, they will be summarized as 

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median (IQR). Mean differences and 95% CIs will be calculated for all continuous variable 
comparisons. Proportion differences and 95% CIs will be calculated for all categorical 
variable comparisons.  
 
16.4.1 Neurocognitive outcomes 
Mean MDAT sub-domain z-scores will be compared across all pairs using Student’s t 
test. Saccade eye-tracking assessment score will be compared across all pairs using 
Student’s t test. Differences with 95% CIs will be reported.  
 
16.4.2 Biochemical outcomes 
Fatty acid percentages will be assessed for distribution shape. If they do not appear to 
approximate the normal distribution, they will be compared using Wilcoxon rank sum 
test; if they do approximate the normal distribution, they will be compared using 
Student’s t test.  
 
16.4.3 Programmatic outcomes 
Chi-square will be used to compare proportions who have recovered, remained 
malnourished, died, and defaulted between DHA-HO-RUTF and S-RUTF, and 
between HO-RUTF and S-RUTF. Differences with 95% CIs will be reported.  
 
16.4.4 Anthropometric outcomes 
Rates of gain in weight, MUAC, and length will be compared using Student’s t test. 
Differences with 95% CIs will be reported.  
 
16.4.5 Analysis by intervention component 
Linear regression will be performed to compare the effects of HO and DHA on 
MDAT global z-scores. Beta coefficients with 95% CI will be reported. Ordinal 
logistic regression will be performed to compare the effects of HO and DHA on the 
PSA composite intention score. ORs and 95% CI will be reported.  
17 Safety Analyses 
There were no predicted safety issues with either of the experimental fatty acid formulations 
of RUTF used in this study. Following study completion, we will compare adherence, 
defaulting, and symptoms, including diarrhea and fever, between DHA-HO-RUTF and S-
RUTF, and between HO-RUTF and S-RUTF. 
18 Quality assurance of statistical programming 
Windows operating system will be used for statistical analysis. Data will be stored in locked 
Microsoft access databases until analysis. Data cleaning will be performed using Microsoft 
Excel and SPSS. At the time of writing, we plan to perform analyses using R and SPSS. All 
R syntax will be dated, saved, and stored.  
19   Attachments 
19.1 Consent document 

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Page 45 of 57 
 
 
 
COLLEGE OF MEDICINE 
Principal  
 
 
 
 
 
 
 
 
 
 
  College of Medicine 
M. H. C. Mipando MSc PhD 
 
 
 
 
 
 
 
 
 
        Private Bag 360 
Chichiri 
Blanytre 3 
Our Ref:  
 
 
 
 
 
 
 
 
 
 
 
     Malawi 
               Telephone:  01 871911 
Your Ref:  
 
 
 
 
 
 
 
 
 
 
 
01 874107 
         Fax: 01 874 700 
 
“Randomized, double-blind, placebo-controlled trial to evaluate the effectiveness of different fats in 
food in the community-based treatment of severe acute malnutrition in Malawian children” 
 
Dr. Ken Maleta  
 
Telephone +265-888-232-202 
 
Your child is invited to participate in a research study conducted by Dr. Maleta and colleagues. 
 
1. The overall purpose of this research is to see if your child will grow and gain weight while 
achieving a more normal neurocognitive status when using therapeutic foods with different fats as 
he/she recovers from severe malnutrition. 
 
2. You will be given one of three peanut butter foods to feed him/her at home.  One of these foods is 
the standard RUTF, the other two RUTFs are experimental.  They contain all of the same nutrients 
that are recommended by WHO, and include some different fats.  One of the fats is from a different 
variety of peanut and the other from fish oil.  You will be given enough food to feed the child for 2 
weeks, and asked to return every 2 weeks to be weighed, measured and given more food.  You will be 
asked to feed your child this food until his/her weight has returned to what is considered normal for 
the child’s height.  
 
After 4 weeks of treatment a small blood sample, 1.5 mL will be taken from his/ her arm to measure 
the amount of essential fatty acids in the body. 
 
After 6 weeks, if your child is one of the following ages, 10-11 mo, 14-15 mo or 18-19 mo he/ she 
will be asked to participate in a special test.  The test has two parts and takes about 30 minutes total.  
The first part of the test is conducted as you sit with your child in your lap watching a computer 
screen.  The computer will show your child some pictures and determine how long the child looks at 
any one picture and how quickly your child looks at new pictures.  The second part of the test 
involves showing your child an interesting object that is enclosed in a plastic box with a hole in it.  
Your child will try to remove the object from the box with his/ her hand.  This exercise will be 
recorded by a video camera.  Experts will review these videos and determine how well your child was 
able to remove the interesting object. Both of these exercises are known to be safe.  
 
A 6 months after recovery and when your child reaches about 3 years of age, he/ she is asked to return 
to participate in a testing exercise in which the child is asked to complete certain small tasks and solve 
certain small problems.  This test takes about 30-60 minutes. Three types of tests will be conducted 
namely: pattern analysis which involves a set of form-board problems testing visual-constructional 

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abilities; copying which involves a set of block building problems also testing visual-constructional 
abilities and bead memory involving a set of visual memory tests.  
 
Your (your child’s) participation in this study is expected to last 8 weeks of therapy.  
 
 
Other malnourished children may benefit because we may learn whether these different therapeutic 
foods are necessary to promote full nutritional recovery from severe malnutrition. 
 
6. Other than non-participation in the research, there are no alternatives.  If you decide not to 
participate, your child will still receive the peanut butter food without any extra fats to help him/her 
recover from the malnutrition. 
 
7. All reasonable measures to protect the confidentiality of your child’s medical records. There is a 
possibility that your child’s medical record, including identifying information, may be inspected and 
photocopied by government or University officials or members of the Human Studies Committee. 
 
8.  You will receive soap at every 2-week follow-up visit and a chitenje upon graduation. 
 
9. If you have any questions or concerns regarding this study, if any problems arise, or there any 
feelings of pressure to participate, you may call the Principal Investigator Dr. Maleta at College of 
Medicine, Blantyre on 0888 232 202. You may also ask questions or state concerns regarding your 
(your child's) rights as a research subject to Dr. YB Mlombe at COMREC’s secretariat, College of 
Medicine on 01 871 911. 
 
10. University of Malawi investigators and their staffs will try to reduce, control, and treat any 
complications from this research.  If you feel that you are injured because of the study, please contact 
the Investigator and/or the Human Studies Committee Chairman.  
 
11. You (your child) will be informed of any significant new findings developed during the course of 
participation in this research that may have a bearing on your (your child's) willingness to continue in 
the study.  The investigator may withdraw you (your child) from this research if circumstances arise 
(such as non-compliance with the protocol and non-tolerance of a study food) which warrant doing so.  
 
12. This research is not intended for the purpose of diagnosing or treating any medical problems not 
specifically stated in the purpose of the research.   
 
I have heard this consent and have been given the opportunity to ask questions.  I will also be given a 
copy of this consent form for my records.  I hereby give my permission (or give permission for my 
child) to participate in the research described above,  
 
Parent’s name: _____________________Parent’s signature/thumbprint:__________
 
Date:________ 
 
Name of child ___________________ 
 
Witness’ name: _____________________Witness’ signature/thumbprint:__________
 
Date:________ 
 
Study staff’s name:___________________study staff’s signature:_________________
 
Date:________ 
 
 

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19.2 WHZ charts 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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19.3 MDAT items sheet 
GROSS MOTOR 
1. Lifts chin up off the floor for 
a few seconds* 
 
2. Prone (on his or her tummy), can 
lift head up to 90 degrees* 
 
3. Lifts head, shoulders and chest 
when lying on stomach* 
4. Holds head upright for a few 
seconds* 
 
5. Pulls to sit with no head lag* 
 
6. Sits with help (with a nice 
straight back)* 
 
7. Holds head erect 
continuously 
when sitting.* 
 
8. Bears weight on legs (holds legs 
strongly when put in standing 
position)* 
 
9. Rolls over from back to front* 
10. Sits by self well.** 
 
11. Crawls (in any way)** 
 
12. Pulls self to stand** 
 
13. Able to stand if holding on 
to things** 
 
14. Walks using both hands of 
someone.  
 
15. Walks with help using someone’s 
hand or furniture  
16. Walks but falls over at times 
 
17. Stoops over and gets back up 
 
18. Walks well 
 
19. Runs (basic running) 
 
20. Kicks a ball (in any way- even only 
a little bit)  
 
21. Runs well, stopping and starting 
without falling over 
22. Does your child climb on to 
furniture to reach things that they 
want? 
 
23. Throws a ball overhand (raising 
his arm and shoulder) 
24. Kneels and can get up 
without using hands.  
25. Climbs and jumps off 
platform with support 
26. Jumps off platform on their own  
27. Throws a 
ball in to a 
basket at 1 
metre  
 
28. Runs, stops 
and kicks a 
ball at some 
distance 
 
29. Jumps with feet together off the 
ground 
 
30. Jumps over 
line on the 
ground 
 
31. Stands on 
one foot for 
more than 1 
second  
 
32.Walks on heels 
six steps  
 
33. Jumps over a piece of 
paper or larger object.  
34. Walks on tip 
toes six steps.  
 
35. Hops on one 
foot four steps 
 
36. Stands on one foot for more 
than 5 s  
 
37.  Can throw a ball up in the 
air and catch it with 2 hands. 
 
38. Heel/toe walk with one foot behind 
the other along the string/same line 
with good balance. 
 
 
 
 
*Not included in questionnaire for this study      ** skipped automatically because of program 
FINE MOTOR & PERFORMANCE 

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1. Follows mum/carer’s 
face to mid line. * 
 
2. Fixes and follows 
completely  
from right to left* 
 
3.Puts hands together in front of 
eyes or mouth. * 
 
 4.Reaches for a large thing.* 
 
5. When holding objects 
tends to put them in the 
mouth* 
 
6. Grasps hold of large 
things.* 
 
7. Can pick a larger object from the 
ground such as a 
wooden spoon.* 
8. Looks at a small object  
held in the hand such as a 
 bean or maize 
piece. * 
 
9. Transfers objects from one hand 
to another* 
10. Picks up small things with 
four fingers in a raking 
motion** 
 
11. Strikes one object with 
another or claps toys or 
hands together** 
 
12. Finds object hidden under 
a sheet** 
 
13. Can use a neat pincer grasp to pick 
up object between thumb and 
forefinger** 
 
14. Puts blocks or stones in and  
out of a plastic tea cup 
in imitation. 
 
15. Copies pushing a little wooden 
or wire car along 
 
16. Puts blocks or 2 cm size 
stones in and out of a plastic 
jar in imitation. 
 
17. Dumps blocks out of 
jar purposefully 
 
18. Scribbles on paper with 
chalk or on the ground with a 
stick in straight lines. 
 
19.Scribbles on paper with chalk or on 
the ground with a stick in a circular 
motion 
20.. Can build a tower of two 
 bricks 
   
21. Puts pegs in a board in a 
longer time.    (< 2 min) 
 
 
 
 
22. Ask child to hold four 
bricks in their hands and to 
give you one of them (make 
sure they have four in their 
hand at the time) 
23. Makes a tower of four 
bricks 
 
 
24. Makes a  
tower of at least six bricks 
25. A Fill up two cups. One with very 
little water and one with a lot of water 
and ask the child to give you the cup 
with more water (do it three times)  
26 Can do the peg board quicker – 
within 30 s (<30s) 
27. Unscrews and screws the cap 
on and off a peanut butter plastic 
jar 
 
28. Threads six beads 
 
 
29. With two cups of 
different sizes (same 
colour) child is able to give 
you bigger cup. Say to the 
child, “Give me the bigger 
one?”  
30. Imitates a vertical line 
drawn by the assessor 
31. Picks the “longer” stick 3 times out 
of 3 tries 
32. Picks the heaviest 
of 
 two objects 3 times 
out of 3 tries 
 
33.Can make a bridge 
34. Makes a doll or a car out of 
clay 
35. Copies a circle drawn 
on paper or on the sand 
 
36. Copies a cross drawn on 
paper or on the sand 
 
37. Can fold a piece of paper in half 
38. Can draw a square 
39. Can make a bridge with 6 
blocks   
40. Can make stairs with 6 
blocks 
41.Can copy a pattern with 
4 bottle tops
 
 
42. Can copy a square pattern 
with 4 bottle tops 
 
*Not included in questionnaire for this study      ** skipped automatically because of program 
 
LANGUAGE / HEARING 
•  •  •  • 
• • • •
•  •  •  • 
•  •  •  • 

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1. Startles to sounds* 
 
2. Happy making sounds such as 
“uh”, “eh”, “a” which are 
separate from crying sounds.* 
3.Laughs and chuckles* 
 
4. Turns to voice – looks in the direction 
of a voice or a sound. * 
 
5. Makes single syllable sounds such 
as .. ma/da/pa/ba* 
6. Responds and turns to his or her 
name being called. 
 
7. Says two syllable babble 
such as ....dada, lala, mimi, 
tata,  
8. Understands when being told 
“no” or being 
cautioned  
 
9. Shakes head or does something 
to indicate “No” 
 
10. Follows one stage commands such as 
“give me the cup” 
11. Unclear talk or jabber in 
sentences..(sounds like sentences but 
not always clear to the listener) 
12. Says two words other than mama 
or dada e.g. cup/cow/spoon/   
 
13. Says two words together 
such as “mama-cup” or 
“dada-water” or “dada 
goes” 
 
 
14. Says at least six or more 
words, but words other than those 
used for mother and father 
15. Follows two stage commands 
“go and get the cup over there and 
put it in the basket” 
 
16. Can pull out/identify from the box up 
to 5 things e.g. cup/ball/spoon/bottle/cloth 
Say to the child “give me the 
cup...ball...spoon...bottle..cloth.”More 
than 5 recognised 
17. Speaks clearly in sentences that 
are understood well (three words 
together at a time).  
18.  Points to more than one body part 
Ask...” Where is your.... 
nose/eyes/mouth”... 
> 1   body part 
 
19. Names at least five (5) 
things in the box. Ask the 
child.... “What is this?” and 
pull out of basket a 
cup/spoon, ball, bottle, 
cloth...  
Named at least 5. 
20. Knows his or her first name – 
When asked, “What is your 
name?” – can tell you their name” 
 
21. Knows actions of objects. 
“Which one is used for sweeping?” 
“Which one is used for 
drinking?” .... Ask them to pick 
them up. 
 
22. Can pull out/identify from the box up 
to 10 things e.g. 
cup/ball/spoon/bottle/cloth/soap/ 
brush/bicycle/plate 
Say to the child “give me the 
cup...ball...spoon etc..etc..” 
More than 10 RECOGNISED 
23. Names at least ten (10) things in 
the box. Ask the child.... “What is 
this?” and pull out of basket a 
cup/ball/spoon/bottle/cloth/ battery/ 
brush/bicycle/plate 
NAMED at least 10 
24. Can the child talk or explain things 
that happened in the past e.g talk to 
you in the past tense about what 
happened yesterday 
25. Can the child sing songs 
or repeat rhymes from 
memory 
26. Is able to categorise 
things...“Tell me some things that 
you eat”...“Tell me some animals 
you know”Can answer at least one 
of these questions 
27 Able to follow a three stage 
command e.g. “stand up, clap your 
hands and go over to the....”.?  
28. Knows the use of objects e.g. “What 
do you do with a cup?” “What do you do 
with soap?” 
29.  Can copy 2 syllables that are 
repeated. Say to the child...When I 
say this…Copy me: 
“Pa, Chi, Tu, Go” 
2 out of 4 eg. Pa, Chi 
30. Knows the answer of 2 out of 3 
questions with an adjective e.g What 
do you do when you are hungry? 
What do you do when you are tired? 
What do you do when you are thirsty? 
31. Understands the term 
“faster”. E.g. “which goes 
faster – a car or a bicycle?” 
32. Can copy 4 syllables that are 
repeated…Say to the child...When 
I say this....copy me.. : “Pa, Chi, 
Tu, Go”4 out of 4______ 
33. Understands prepositions e.g. 
“Put the stone/block under the cup, 
put the stone/block on the cup/put 
it next to the cup/put it behind the 
cup”. Needs to do 3 of 4 of these.  
34. Knows opposites e.g. “A man is big, a 
baby is .....”, “If the sun comes up in the 
day, the moon comes up in......” (must do 
2 of 3 of these) 
35 Knows quantities…can count 
some bricks or stones...Put bricks on 
the table and ask the child...” Can 
you count these?” Can count up to 
THREE bricks 
36 Knows quantities...can count some 
bricks or stones...  
Put bricks on the table and ask the 
child.... “Can you count these?” Can 
count up to FIVE bricks 
37. Knows quantities – can 
count up to TEN (10) bricks 
or stones 
38. Knows materials eg. What is a 
cup made of (plastic), a stick 
(wood), a wire bicycle (wire), a 
book (paper). 2 of 3 answered. 
39. Child knows how old they are. 
Can answer the question: How old 
are you?  
40. Child can tell you where he or she 
currently lives e.g. the village or place 
 
 
*Not included in questionnaire for this study       
 

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PERSONAL-SOCIAL 
1. Smiles but not at a particular 
person. * 
 
2. Smiles in response to a person.* 
 
3. Frolics with mother or 
caregiver in response to being 
played with.* 
 
4. Frolics alone, happily playing, 
moving around 
moving body and 
kicking legs in a 
happy way.* 
 
 5.Recognises or settles (quiets) 
with caregivers/known family 
members. Stops crying or quiets 
when mum or another known 
carer takes the baby.* 
 6. Will take phala (porridge)  from a spoon 
when fed by a caregiver* 
 
 7.Helps hold a cup while mum 
gives a drink.* 
  
 
 8. Stretches to be picked up.  
 
 9. Can HOLD a spoon with 
phala (porridge) but NOT get 
to mouth well yet. 
 
 10. Drinks from a cup 
by self without 
spilling.  
 
11. Is able to indicate by pointing 
that they want 
something. 
 
12. Can eat by picking up morsels (of 
nsima/maize) made by mum.   
 13. Puts hands out to be washed 
by mum or carer – helps by 
putting hands out. 
 
14. Can hold a spoon and take phala 
(porridge) by self but spills some/a 
little bit 
 
 15.Indicates in some way the 
need for a poo or a pee. For 
example by crying, pulling at 
pants or saying something... 
16. Wants to join in singing games. 
Ask “Does the child like to be 
included in singing games even if 
he/she  can not yet do them?” 
 
17. Able to greet either by 
extending hand or verbally.  
 
18. Understands to share things with others 
and will do so if with friends or family and 
asked to share.  
 
19. Does a poo or a pee by 
themselves without wetting their 
pants.  
 
  20. Eats phala (porridge) off a 
spoon without spilling.   
 
21. Does the child play by 
pretending objects are 
something else?  
22. Will the child pretend to cook or 
make something (like porridge) with 
imaginary play things e.g tin and 
sand?  
 23. Can make own morsels of 
nsima/maize and put in mouth 
(often with relish) 
 
 
24. Is able to undress by themselves – can 
take off even one item of clothing 
e.g. shorts or skirt..need to be able 
to remove it completely. 
     
25. Wants to go and visit a friend’s 
house… shows independence 
26.Can go to the toilet by themselves 
anywhere (not necessarily at the pit 
latrine)  
 27. Can eat food or relish with 
bits or bones in it such as fish 
with bones or tangerines with 
seeds 
 
 
 28. Is able to dress but not 
completely. Can put on at least one 
item e.g. T-shirt/skirt 
 
29. Washes hands well by self 
before/after eating 
 
 
30. Knows how to keep quiet at important 
meetings or ceremonies. 
 
31.Does household chores in a 
useful way e.g. drawing water or 
hoeing. could be even a small 
amount of help but not just 
pretend. 
 
 32. Able to dress by self completely. 
May need help with buttons, shoe, 
laces or zips. 
 
33. Understands the concept of 
discipline e.g. causes and 
consequences such as that 
saying bad words might lead to 
punishment... 
 
34. Plays games with turn taking 
such as fulaye, mira, jingo.  
 
 
35. Knows how to be respectful to 
elders. Is polite and shows 
respect such as putting hands 
together or kneeling before elders 
 
36. Can go to the toilet or pit latrine by self.  
 
*Not included in questionnaire for this study   

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