Improving Estimates of the Global Burden of Thiamine Deficiency Disorders and Approaches to Their Control: Trial of Thiamine Supplementation in Cambodia
Phase: NA
Conditions: Thiamine Deficiency
Sponsor: Mount Saint Vincent University

Trial of thiamine supplementation in Cambodia: Protocol 
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PROTOCOL 
 
 
 
 
Study Title: 
Trial of thiamine supplementation in Cambodia 
 
Version:  
Version 3.0. August 14, 2019. 
 
Funding:  
Bill & Melinda Gates Foundation; Sackler Institute for Nutrition 
Science, New York Academy of Sciences (OPP1176128_2017; 
Objective 3) 
 
Principal Investigator: 
Kyly C Whitfield, PhD 
 
 
 
Assistant Professor, Department of Applied Human Nutrition 
 
Mount Saint Vincent University 
166 Bedford Hwy, Halifax, Nova Scotia, B3M 2J6, Canada 
 
 
 
Phone: +1 (902) 457-5978 
 
 
 
Email: kyly.whitfield@msvu.ca 
 
Statement of Compliance 
This document is a protocol for a randomized controlled trial. The study will be conducted in 
compliance with all stipulations of this protocol and the conditions of ethics committee approval. I 
agree that the study will be conducted in accordance with the conditions outlined in the protocol 
(subject to any amendments). I have read and understood the protocol.  
 
KYLY C WHITFIELD 
 
 
 
Investigator’s name 
 
 
 
 
 
____________________________________ 
 
August 14, 2019.           . 
Investigators signature  
 
 
 
 
Date 

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Table of Contents 
 
Statement of Compliance .............................................................................................................................. 1 
1. Study Synopsis ............................................................................................................................................. 4 
1.1 Title ......................................................................................................................................... 4 
1.2 Objectives .................................................................................................................................. 4 
1.3 Design ...................................................................................................................................... 4 
1.4 Outcomes and Analysis Objectives ................................................................................................. 4 
1.5 Study Duration .......................................................................................................................... 4 
1.6 Interventions .............................................................................................................................. 4 
1.7 Sample size ............................................................................................................................... 5 
1.8 Population ................................................................................................................................ 5 
2. Investigators, Scientific Advisory Board, and Study Locations .................................................... 5 
2.1 Study investigators ...................................................................................................................... 5 
2.2 Statisticians ............................................................................................................................... 6 
2.3 Scientific Advisory Board ............................................................................................................. 6 
2.4 Study Location........................................................................................................................... 7 
2.5 Study Management ..................................................................................................................... 7 
2.6 Serious Adverse Events Committee / Data Safety Monitoring Board ................................................... 7 
2.7 Funding .................................................................................................................................... 7 
3. Introduction, Background, and Rationale .......................................................................................... 7 
3.1 Thiamine, and thiamine deficiency .................................................................................................. 7 
3.2 Dietary thiamine requirements ....................................................................................................... 8 
3.3 Biomarkers of thiamine status ....................................................................................................... 8 
3.4 Efforts to improve perinatal thiamine intake .................................................................................... 9 
3.5 Thiamine fortification .................................................................................................................. 9 
3.6 Salt as a fortification vehicle .......................................................................................................... 9 
3.7 Summary of rationale .................................................................................................................. 9 
4. Study Objectives ........................................................................................................................................ 10 
5. Study Design .............................................................................................................................................. 11 
6. Study Treatments ...................................................................................................................................... 11 
6.1 Description of study arms ........................................................................................................... 11 
6.2 Treatment arm codes and packaging of study capsules ....................................................................... 12 
6.3 Safety of thiamine doses .............................................................................................................. 12 
6.3 Product accountability ................................................................................................................ 12 
6.4 Compliance ............................................................................................................................. 13 

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7. Sample Size ................................................................................................................................................. 13 
8. Enrolment and Randomization ............................................................................................................ 14 
8.1 Recruitment ............................................................................................................................. 14 
8.2 Eligibility criteria ..................................................................................................................... 14 
8.3 Randomization procedures and blinding ........................................................................................ 14 
8.4 Breaking of the study blind ......................................................................................................... 15 
8.5 Participant withdrawal .............................................................................................................. 15 
9. Study Procedures ...................................................................................................................................... 15 
9.1 Chart review ............................................................................................................................ 16 
9.2 Fortnightly monitoring ............................................................................................................... 16 
9.3 Salt disappearance .................................................................................................................... 16 
9.4 Questionnaire .......................................................................................................................... 17 
9.5 Anthropometrics ....................................................................................................................... 17 
9.6 Venous blood samples ............................................................................................................... 17 
9.7 Human milk samples ................................................................................................................ 17 
9.8 Blood and human milk processing and analysis ............................................................................... 17 
9.9 Cognitive assessments ................................................................................................................. 19 
9.10 Participant remuneration .......................................................................................................... 20 
10. Adverse Event Reporting ...................................................................................................................... 20 
11. Statistical Analysis ................................................................................................................................... 21 
12. Administrative Aspects, and Data Security and Management .................................................. 21 
12.1 Obtaining informed consent ....................................................................................................... 21 
12.2 Ethics .................................................................................................................................. 22 
12.3 Confidentiality and data security ................................................................................................ 22 
12.4 Modifications to the protocol ...................................................................................................... 22 
13. Use of Data, and Dissemination ........................................................................................................ 23 
13.1 Layperson dissemination .......................................................................................................... 23 
13.2 Academic dissemination ........................................................................................................... 24 
14. References ................................................................................................................................................. 25 
Appendices ...................................................................................................................................................... 29 
 

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1. Study Synopsis 
 
1.1 Title 
 
Trial of thiamine supplementation in Cambodia 
 
Note that this study was funded using the title: ‘Improving estimates of the global burden of 
thiamine deficiency disorders (TDDs) and approaches to their control - Objective 3: Trial of 
thiamine supplementation in Cambodia.’  
 
1.2 Objectives 
 
1. To conduct a dose response supplementation trial among lactating Cambodian women to 
determine the lowest dose of thiamine that yields the maximum increase in human milk 
thiamine.  
2. To assess rural Cambodian households’ usual salt intake using a household salt disappearance 
study.  
3. To assess cognitive development of infants aged 2 weeks – 12 months to mothers consuming 
varying thiamine doses using a small battery of cognitive assessments.   
 
1.3 Design 
 
Double-blind, four-parallel arm, placebo-controlled randomized trial. 
 
1.4 Outcomes and Analysis Objectives 
 
The primary outcome of this study is human milk total thiamine concentration at 24 weeks 
postpartum. The primary objective of this study is to estimate the dose on the dose response curve 
where additional maternal intake of thiamine (oral dose) no longer meaningfully increases human 
milk total thiamine concentration at 24 weeks postpartum. A full list of outcomes and analysis 
objectives can be found in Section 4.  
 
1.5 Study Duration 
 
This is a two-year study, to be completed between 2018 and 2020. The intervention is 22 weeks, 
from 2 weeks through 24 weeks postnatal, with an additional follow-up at 12 months.  
 
1.6 Interventions 
 
Participants will be randomized to one of four treatment arms, and will be asked to consume one 
oral capsule daily. Treatment arms are:  
• negative control group (placebo; 0 mg thiamine) 
• EAR group (1.2 mg thiamine as thiamine hydrochloride) 
• double EAR group (2.4 mg thiamine as thiamine hydrochloride) 
• positive control group (10 mg thiamine as thiamine hydrochloride) 
 

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1.7 Sample size 
 
320 women and their newborn infants. 
 
1.8 Population 
 
Participants must meet the following criteria: 
• mothers of a newborn 
• aged 18-45 years 
• most recent pregnancy was normal (i.e. no known chronic conditions, preeclampsia, 
gestational diabetes etc.), and the singleton infant was born without complications (e.g. low 
birth weight (<2.5 kg), tongue tie, cleft palate) 
• intends to exclusively breastfeed for six months 
• resides in Kampong Thom province, Cambodia, and is not planning to move in the next six 
months 
• is not currently taking, and hasn’t taken any thiamine-containing supplements over the 
previous 4 months 
• is not currently participating in any nutrition programs beyond normal care 
• is willing to consume one capsule daily from 2 weeks through 24 weeks postpartum 
• is willing for her entire household consume only salt provided by the study team 
• is willing for the following biological samples to be collected: a maternal venous blood 
sample and human milk sample at 2 weeks postpartum, a human milk sample at 4 and 12 
weeks postpartum, and maternal and infant blood samples and a human milk sample at 24 
weeks postpartum. 
 
2. Investigators, Scientific Advisory Board, and Study Locations  
 
2.1 Study investigators 
 
Kyly C Whitfield 
Principal Investigator 
Department of Applied Human Nutrition 
Mount Saint Vincent University 
Halifax, Nova Scotia, Canada 
kyly.whitfield@msvu.ca  
 
Hou Kroeun 
Co-Investigator 
Helen Keller International, Cambodia 
Phnom Penh, Cambodia 
khroeun@hki.org  
Keith Porter 
Co-Investigator 
Helen Keller International, Cambodia 
Phnom Penh, Cambodia 
kporter@hki.org 
 
Tim J Green 
Co-Investigator 
Healthy Mothers, Babies and Children Theme 
South Australia Health and Medical Research 
Institute 
Adelaide, South Australia, Australia 
tim.green@sahmri.com  
 
 
 

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Frank Wieringa  
Co-Investigator 
Institut de recherche pour le développement 
Montpellier, France 
franck.wieringa@ird.fr 
 
Mam Borath 
Co-Investigator 
National Sub-Committee for Food Fortification 
Cambodia Ministry of Planning 
Phnom Penh, Cambodia 
borathmam.mop@yahoo.com 
 
Sophonneary Prak 
Co-Investigator 
National Nutrition Programme 
Cambodia Ministry of Health 
Phnom Penh, Cambodia 
sophonprak@gmail.com 
  
Jeff Measelle 
Co-Investigator 
Department of Psychology 
University of Oregon 
Eugene, Oregon, USA 
measelle@uoregon.edu   
Dare Baldwin 
Co-Investigator 
Department of Psychology 
University of Oregon 
Eugene, Oregon, USA 
baldwin@uoregon.edu  
Geraldine Richmond 
Co-Investigator 
Department of Chemistry and Biochemistry 
University of Oregon 
Eugene, Oregon, USA 
richmond@uoregon.edu  
 
 
2.2 Statisticians 
 
Lisa Yelland 
Statistician 
South Australia Health and Medical Research 
Institute 
Adelaide, South Australia, Australia 
lisa.yelland@adelaide.edu.au  
Shalem Leemaqz 
Statistician 
South Australia Health and Medical Research 
Institute 
Adelaide, South Australia, Australia 
shalem.leemaqz@sahmri.com  
 
2.3 Scientific Advisory Board 
 
Megan Bourassa 
Sackler Institute for Nutrition Science 
New York Academy of Sciences 
New York, New York, USA 
mbourassa@nyas.org  
Levente Diosady 
Chemical Engineering & Applied Chemistry 
University of Toronto 
Toronto, Ontario, Canada 
l.diosady@utoronto.ca  
 
Lisa Houghton 
Department of Human Nutrition 
University of Otago 
Dunedin, New Zealand 
lisa.houghton@otago.ac.nz  
Annie Wesley 
Agriculture and Food Security  
International Development Research Centre 
Ottawa, Ontario, Canada 
awesley@idrc.ca  
  
 
Arnaud Laillou 
UNICEF Cambodia 
Phnom Penh, Cambodia 
alaillou@unicef.org 

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2.4 Study Location 
 
Households in Kampong Thom province, Cambodia within the catchment area of the following 
Health Centres: Tboung Kapoeur, Kampong Svay, Sankor, Chey, Salavisai, Prey Kuy, Prey Pros, and 
Srayov Health Centres. 
 
2.5 Study Management 
 
The Principal Investigator and Co-Investigators will oversee the coordination of the study; they will 
meet bi-monthly, or more often if required. Helen Keller International, Cambodia will be the 
primary implementer of the study, overseeing day-to-day operations, including recruitment, consent, 
data and biological sample collection, and data management. The Scientific Advisory Board will 
meet with the investigators annually. 
 
2.6 Serious Adverse Events Committee / Data Safety Monitoring Board  
 
There is no necessity for a Serious Adverse Events Committee or a Data Safety Monitoring Board 
(DSMB) in the current study because of the absence of potential adverse effects. There is no 
tolerable upper intake level (UL) for thiamine because there has never been an adverse event 
reported from high thiamine intake (1,2), even though over-the-counter supplements in Canada and 
the United States commonly contain 50 mg of thiamine or more. Hence, we are confident that there 
is a very low risk of serious adverse events in this study related to the intervention.  
 
However, we do expect some infant deaths in our cohort due to other causes. As per the 2014 
Cambodian Demographic and Health Survey (CDHS), the rate of neonatal (birth to one month) and 
infant (birth to one year) mortality was 29 and 11 deaths per 1,000 live births, respectively (3). The 
majority of neonatal deaths occur in the first 6 days postnatal: in the 4 years preceding the 2014 
CDHS data collection, 82% of neonatal deaths occurred in the first 6 days after birth (3). Since this 
study starts at 2 weeks postnatally, we would expect the mortality rate in our study to lie between 
1.67 (neonatal mortality > 6 days postnatal for Kampong Thom = 0.029*0.18*320) and 3.68 (infant 
mortality in Kampong Thom = 0.011*320) infant deaths.  
 
2.7 Funding 
 
This study is funded through the Bill & Melinda Gates Foundation and the Sackler Institute for 
Nutrition Science, New York Academy of Sciences (Opportunity ID OPP1176128) as ‘Objective 3: 
Trial of thiamine supplementation in Cambodia’, as part of the larger program grant entitled 
‘Improving estimates of the global burden of thiamine deficiency disorders (TDDs) and approaches 
to their control’. The cognitive follow-up assessment at 12 months postnatal is funded by the New 
York Academy of Sciences.  
 
3. Introduction, Background, and Rationale 
 
3.1 Thiamine, and thiamine deficiency 
 
Thiamine (vitamin B1) is an essential water-soluble micronutrient required for energy metabolism (4). 
Thiamine deficiency remains an under-studied public health issue, recently being called the forgotten 
disease of Asia (5). There is evidence of suboptimal thiamine intake or status throughout Southeast 

Trial of thiamine supplementation in Cambodia: Protocol 
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Asia, notably in Cambodia (6–11), Laos (5,12–14), and among Karen refugees during their early 
arrival in the Mae La Camp on the Thai-Myanmar border (15,16).  
 
Infantile beriberi, a disease caused by thiamine deficiency, presents during the exclusive 
breastfeeding period and without treatment can result in death within hours of clinical presentation 
(15). The dietary staple of Cambodia is B-vitamin poor, white, polished rice; it makes up upwards of 
60% of daily energy intake (17). Women with poor dietary thiamine intake produce milk low in 
thiamine, putting their exclusively breastfed infants at a high risk of developing infantile beriberi 
(18). We recently demonstrated that maternal consumption of a thiamine-fortified fish sauce 
significantly increased maternal, human milk, and infant thiamine status (10). 
 
There is also a growing body of evidence suggesting thiamine deficiency not severe enough to cause 
clinical symptoms has a negative effect on cognitive development and functioning (19–22), though 
the precise pathways and mechanisms linking these two factors remain poorly understood. 
Important questions remain about the timing of deficiency, levels of deficiency, and the ability of 
prophylactic interventions to prevent or therapeutic interventions to remediate the effects of 
suboptimal thiamine status on cognitive outcomes in humans (23).  
 
3.2 Dietary thiamine requirements 
 
The Institute of Medicine’s Dietary Reference Intakes (DRI) can be used to assess dietary thiamine 
adequacy (24). Thiamine has an estimated average requirement (EAR), the median requirement for a 
given lifestage group, and a statistically derived recommended dietary allowance (RDA), an estimate 
of the daily average dietary intake that meets the nutrient needs of 97.5% of healthy members of the 
group (24). There is no UL for thiamine because there have been no reports of adverse effects of 
excess thiamine intake (1,2,25). Lactating women secrete an estimated 0.16 mg thiamine per day into 
human milk, and additional thiamine is required by the mother herself to meet increased energy 
needs for milk production (1). For lactating women, the EAR and RDA are 1.2 and 1.4 mg/day, 
respectively (1).  
 
For infants, an adequate intake (AI) was derived due to insufficient evidence to set an EAR and, in 
turn, a RDA (1). The thiamine concentrations of milk from healthy, well-nourished mothers are 
extrapolated, assuming a daily milk intake of 780 mL (1). The thiamine AI for infants aged 0-6 
months is 0.2 mg/day, however, the evidence base used for setting this AI is poor due to the small 
sample size and the outdated analytical techniques to assess human milk thiamine concentrations 
(26). 
 
3.3 Biomarkers of thiamine status 
 
Two biomarkers are used to assess thiamine status. Thiamine diphosphate (ThDP) is the biologically 
active form of the vitamin (4), and concentrations can be assessed in whole blood or erythrocytes 
using high performance liquid chromatography with a fluorescence detector (27). Unfortunately 
there is currently no consensus on the most appropriate ThDP cut-offs for thiamine deficiency (28). 
The other assessment method is a functional marker of thiamine status, transketolase activity 
coefficient. ThDP is a cofactor with transketolase in the pentose phosphate pathway; in vitro 
measurement of the activity of transketolase before and after the addition of excess ThDP reveals 
the original level of ThDP saturation, indicating thiamine adequacy (1,25). An erythrocyte 
transketolase activity coefficient >1.25 is commonly used as a cut-off for thiamine deficiency (1).  

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3.4 Efforts to improve perinatal thiamine intake 
 
Low maternal thiamine intake reduces the thiamine concentration of human milk (18). Infantile 
thiamine deficiency and beriberi are not normally seen in countries where maternal thiamine intake 
is adequate (29). Both supplementation and fortification interventions have been employed in an 
attempt to improve maternal thiamine status as a means of preventing infantile thiamine deficiency.  
Between 1987 and 1990 there was an outbreak of infantile beriberi in the Mae La refugee camp on 
the Thai-Burmese border that accounted for 40% of infant mortality (15,16). As a preventative 
measure, lactating women in this camp were provided with a weekly 10 mg thiamine supplement 
until 9 months postpartum (15). However, supplementation has several challenges, including high 
cost, issues of distribution and a sustainability, and compliance.  
 
3.5 Thiamine fortification 
 
Fortification is a potentially more suitable alternative to supplementation, as it is a sustainable, cost-
effective, and passive intervention (30–33). Fortification is a highly successful public health 
intervention; for example, two thirds of the world’s population has access to iodized salt (34), and 
folic acid fortification in Canada and elsewhere has reduced the incidence of neural tube defects 
(35). Wheat flour is fortified in many countries (36), however this is not appropriate in Cambodia 
and other countries where wheat flour is not a food staple. We recently conducted a randomized 
controlled efficacy trial of thiamine-fortified fish sauce, and found that consumption over 6 months 
improved blood and human milk thiamine concentrations of mothers, their breastfed infants < 6 
months, and children < 5 years (10,11). However, centrally produced fish sauce may not reach the 
poorest communities who make their own fish sauce, and fish sauce consumption is not universal in 
all regions where we find thiamine deficiency (37,38).  
 
3.6 Salt as a fortification vehicle 
 
Salt is a common condiment in most regions of the world, and has proven to be a successful global 
fortification vehicle for iodine (39). Current estimates suggest usual per capita salt intake in 
Cambodia is 15 g/day day (40). However, we do not know how much is consumed at the individual 
level. In addition, the WHO has been actively promoting salt reduction programs globally with a 
goal to decrease salt intake to 5 g/day (41), therefore new measures of salt intake are required.  
 
3.7 Summary of rationale 
 
Suboptimal maternal thiamine intake puts exclusively breastfed infants at risk of low thiamine status, 
impaired cognitive development, and infantile beriberi, which can be fatal. Thiamine fortification of 
salt is a potentially low-cost and sustainable means of combating suboptimal thiamine status, 
however knowledge gaps must be filled before thiamine fortification can proceed. There is limited 
data available on the dose of thiamine required by lactating women to optimize the thiamine 
concentrations in their milk. Further, usual salt intake among lactating women is unknown. Finally, 
although there is emerging evidence that low thiamine intake in early life impacts cognitive 
development, this has yet to be assessed in a controlled research environment. The overall aim of 
this study is to obtain the information necessary to formulate a thiamine-fortified salt for future use 
in Cambodia (ideal dose, usual salt intake), and to explore the impact of various doses of maternal 
thiamine on makers of infant cognitive development.   

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4. Study Objectives 
 
Primary Outcome and Analysis Objective: 
To estimate the dose on the dose response curve where additional maternal intake of thiamine (oral 
dose) no longer meaningfully increases human milk total thiamine concentration at 24 weeks 
postpartum. 
 
Secondary Outcomes and Analysis Objectives: 
1. To estimate the dose on the dose response curve where additional maternal intake of 
thiamine (oral dose) no longer meaningfully increases infant thiamine diphosphate 
concentrations (ThDP) 24 weeks postnatally, and assess whether this depends on the 
presence/absence of a genetic hemoglobin disorder. 
2. To estimate the dose on the dose response curve where additional maternal intake of 
thiamine (oral dose) no longer meaningfully increases human milk total thiamine 
concentration at 4 and 12 weeks postpartum. 
3. To estimate the dose on the dose response curve where additional maternal intake of 
thiamine (oral dose) no longer meaningfully increases infant transketolase activity at 24 
weeks postnatally, and assess whether this depends on the presence/absence of a genetic 
hemoglobin disorder. 
4. To estimate the dose on the dose response curve where additional maternal intake of 
thiamine (oral dose) no longer meaningfully increases maternal ThDP at 24 weeks 
postpartum, and assess whether this depends on the presence/absence of a genetic 
hemoglobin disorder. 
5. To estimate the dose on the dose response curve where additional maternal intake of 
thiamine (oral dose) no longer meaningfully increases maternal ETKac at 24 weeks 
postpartum, and assess whether this depends on the presence/absence of a genetic 
hemoglobin disorder. 
6. To test for differences between the 4 randomised groups on human milk total thiamine at 4, 
12, and 24 weeks postpartum.  
7. To test for differences between the 4 randomised groups on maternal ThDP at 24 weeks 
postpartum, and assess whether this depends on the presence/absence of genetic 
hemoglobin disorder. 
8. To test for differences between the 4 randomised groups on maternal ETKac at 24 weeks 
postpartum and assess whether this depends on the presence/absence of a genetic 
hemoglobin disorder. 
9. To estimate usual household salt intake from mean fortnightly salt disappearance (weight 
lost, in g). 
10. To estimate salt intake among a subset of 100 lactating women, their male partners (if 
applicable), and their children 24-59 months (if applicable) using observed weighed salt 
intake records. 
11. To estimate sodium intake using 24 hr urinary sodium concentrations among a subset of 100 
lactating women. 
12. To test for differences between the 0 & 10 mg randomised groups on Composite Mullen 
and the 5 subscales of the Mullen at 24 and 52 weeks postnatally. 
13. To test for differences between the 0 & 10 mg randomised groups on Visual Paired 
Comparison Novelty Score and the attention and processing speed subscales at 24 and 52 
weeks postnatally. 

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14. To test for differences between the 0 & 10 mg randomised groups on the Language 
Preference Task Score at 24 and 52 weeks postnatally. 
15. To test for differences between 0 & 10 mg randomised groups on oculomotor scores at 24 
and 52 weeks postnatally. 
16. To determine the effect of inflammation, as measured by C-reactive protein (CRP) and α-1-
acid-glycoprotein (AGP) on maternal ThDP at 2 and 24 weeks postpartum, and infant 
ThDP at 24 weeks postnatal. 
 
5. Study Design 
 
This will be a double-blind, four-parallel arm randomized controlled trial, in which 320 women and 
their newborn infants will be enrolled. Each dyad will participate in the study between 2 and 24 
weeks postnatal, a 22-week intervention period. The primary outcome measure is human milk 
thiamine concentrations at 24 weeks postpartum (see Figure 1). There are additional cognitive 
assessments at 12 months (52 weeks) postnatal. 
 
Figure 1: A pictorial description of the study design and data collection scheme. 
 
6. Study Treatments 
 
6.1 Description of study arms 
 
The four study arms are described in Table 1, below. 
 
Table 1: Treatment arms for the trial. 
Treatment Arm 
Rationale 
Thiamine Dose 
Negative Control 
Negative Control (placebo) 
0 mg/d 
EAR 
1 x thiamine EAR for lactating women (1) 
1.2 mg/d 
Double EAR 
2 x thiamine EAR for lactating women (1) 
2.4 mg/d 
Positive Control 
Positive Control (dose currently given in supplemental form 
in Myanmar) 
10 mg/d 
 
Supplements will be opaque capsules containing varying amounts of thiamine hydrochloride and 
cellulose filler, as indicated in Table 1. All thiamine is delivered as thiamine hydrochloride, 
calculated using a 1.271 correction factor (ratio of molecular weights of thiamine hydrochloride and 

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thiamine). Capsules will be formulated and compounded by pharmacist Anastasia Hanias at the 
Quinpool Wellness Centre in Halifax, Nova Scotia, Canada.  
 
6.2 Treatment arm codes and packaging of study capsules 
 
Statisticians at the South Australian Health and Medical Research Institute (SAHMRI) will create a 
total of 8 alpha-numeric codes consisting of 3 letters followed by 8 numbers to label study 
supplements, two sets of codes per treatment arm.   
 
Study capsules will be packaged in 14-capsule blister packs and delivered to the participants every 
two weeks. These blister packs will contain the following labeling in both English and Khmer: 
• “For research purposes only”  
• “Could contain up to 10 mg thiamine (vitamin B1)” 
• “Take one capsule each morning” 
• best before date 
• alpha-numeric treatment code 
• days of the week on each blister slot 
• study ID and barcode (individualized sticker) 
 
6.3 Safety of thiamine doses 
 
The thiamine hydrochloride safety data sheet can be found in Appendix A. The thiamine EAR is 
1.2 mg/day (1), however there is no concern for regular consumption of higher doses because 
thiamine supplements above these doses are commonly available over-the-counter in Canada and 
the United States. For example in Canada, Jamieson Natural Sources has Health Canada approval to 
sell B Complex vitamin supplements containing up to 100 mg each of thiamine. In addition, as 
noted in 3.2 Dietary thiamin requirements, there is no UL for thiamine because there have been no 
reports of adverse effects of excess thiamine intake (1,2,25).  
 
6.3 Product accountability 
 
Initial Assessment 
All capsules will be formulated and packed in two batches at the Quinpool Wellness Centre in 
Halifax, Nova Scotia, Canada, in July and September 2018 using codes assigned by an independent 
scientist not involved in this study. Before data collection, an independent lab will assess a sample of 
capsules from each batch to confirm thiamine concentrations. The independent scientist will courier 
10 capsules from each of the 8 treatment codes to the independent lab for analysis. The lab report 
will be shared with an independent scientist, who will cross-check values with the treatment arm 
codes to ensure the supplements meet the study standards. The supplements will be deemed 
acceptable if the average thiamine concentration for each code (n=10 samples) falls within 15% of 
the values shown in Table 1. 
 
Throughout the study 
A set of supplement blister packs will be stored at the village chief’s home (in a manner similar to 
how women are storing their blister packs) in each of the eight health centre catchment areas in 
which we will work. Field staff will collect one blister pack from each of the village chief’s homes 
every second month for assessment. We will collect 1 blister pack x 8 treatment codes x 8 village 

Trial of thiamine supplementation in Cambodia: Protocol 
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chief’s homes = 64 blister packs will be couriered to the independent lab. Here, the lab technician 
will select 5 capsules from each blister pack for analysis; supplements will be deemed acceptable if 
the average thiamine concentration for each code (n=40 samples) falls within 15% of the values 
shown in Table 1. 
 
6.4 Compliance 
 
Compliance will be assessed fortnightly: field staff will visit the participant’s home to pick up the old 
blister pack and complete a capsule count, and to deliver a new blister pack. A woman will be 
considered compliant if she consumes ≥80% capsules over the 22-week intervention.  
 
In an attempt to ensure high compliance, participants will interact with the same field staff person 
throughout the entire study in hopes of building rapport. This field staff person will complete in-
person check-ins fortnightly throughout the study, and will also make weekly phone calls to 
encourage women to continue consuming their study capsules daily. All participants will be provided 
with a mobile phone as part of this study.  
 
We are also conducting a small compliance pilot study in advance of this randomized controlled trial 
to determine if additional reminders in the form of daily voice messages (e.g. Verboice) or text SMS, 
in addition to the weekly phone calls and fortnightly visits, are warranted. 
 
7. Sample Size 
 
To detect a clinically meaningful difference of 40 μg/L in human milk total thiamine concentration 
between any two treatment groups with 90% power, assuming a SD of 43 µg/L (estimated SD of 
control group in a Cambodian thiamine-fortified fish sauce trial (9)), 48 women are required per 
treatment group, or a total of 192 women. This sample size allows for 20% attrition and uses a two-
sided alpha of 0.0083 for each of the 6 pairwise comparisons between the four treatment groups in 
order to control the familywise error rate at the 0.05 level using a Bonferroni adjustment for multiple 
comparisons. Recruitment of 320 participants (80 per group) was planned to allow for some 
uncertainty in the assumed values, particularly the SD which may be larger than anticipated. 
 
Simulations of 500 dose-response curves for human milk total thiamine concentration were 
conducted to estimate the precision that this sample size would provide for addressing the primary 
study objective (i.e. for estimating the dose required to achieve 90% of the average maximum human 
milk total thiamine concentration). Data were simulated based on an average minimum and 
maximum concentration of 136 µg/L (9) and 210 µg/L (1), respectively, with a SD of 43 µg/L (9), 
and reaching 50% and 90% of the maximum average concentration at dose 1.2 mg/d (EAR group) 
and 2.4 mg/d (double EAR group) respectively. Assuming an Emax dose-response curve, the 
precision of the estimated dose is ±1.29 mg/d (i.e. the 90% confidence interval for the estimated 
dose will be within ±1.29 mg/d of the point estimate). 

Trial of thiamine supplementation in Cambodia: Protocol 
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8. Enrolment and Randomization 
 
8.1 Recruitment 
 
We plan to recruit and enroll the majority of participants through antenatal care (ANC) visits, as well 
as through consultation with local village chiefs, elders, and health centre staff. Pregnant women will 
be advised of the study during ANC programs within the local health centres in Kampong Thom 
province in which we will work. The following health centres were selected in consultation with the 
National Nutrition Programme (Cambodian Ministry of Health) because of their high birth rates: 
Tboung Kapoeur, Kampong Svay, Sankor, Chey, Salavisai, Prey Kuy, Prey Pros, and Srayov Health 
Centres. Local midwifes and nurses will provide a general overview of the research study, and will 
share names and contact information of interested women with the research team. The research 
team will follow up with women after delivery, screen for eligibility, obtain consent, and enroll 
women. Recruitment will continue on a rolling basis until 320 women are enrolled (we anticipate 
rolling recruitment over five months). A record of all women screened but not enrolled will be 
maintained.  
 
8.2 Eligibility criteria 
 
All women must provide written informed consent to participate. Participants must meet the 
following criteria: 
• mothers of a newborn 
• aged 18-45 years 
• most recent pregnancy was normal (i.e. no known chronic conditions, preeclampsia, 
gestational diabetes etc.), and the singleton infant was born without complications (e.g. low 
birth weight (<2.5 kg), tongue tie, cleft palate) 
• intends to exclusively breastfeed for six months 
• resides in Kampong Thom province, Cambodia, and is not planning to move in the next six 
months 
• is not currently taking, and hasn’t taken any thiamine-containing supplements over the 
previous 4 months 
• is not currently participating in any nutrition programs beyond normal care 
• is willing to consume one capsule daily from 2 weeks through 24 weeks postpartum 
• is willing for her entire household consume only salt provided by the study team 
• is willing for the following biological samples to be collected: a maternal venous blood 
sample and human milk sample at 2 weeks postpartum, a human milk sample at 4 and 12 
weeks postpartum, and maternal and infant blood samples and a human milk sample at 24 
weeks postpartum 
 
8.3 Randomization procedures and blinding 
 
Women will be randomly assigned to one of the four treatment groups as described in Table 1. 
Participants, field staff, all study investigators, and data analysts will be blinded to the randomized 
groups. The randomization will be stratified by study centre and use randomly permuted blocks of 
size 8 within strata to assign participants to one of eight treatment codes in the ratio 1:1:1:1:1:1:1:1 (2 
treatment codes per treatment group). An individual who is independent of the trial will determine 

Trial of thiamine supplementation in Cambodia: Protocol 
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which treatment codes correspond to which treatment group. A computer-generated randomization 
schedule will be prepared by the study statisticians using ralloc.ado in Stata. 
 
When a participant is enrolled in the study they will be assigned a unique study ID. The field staff 
will open an envelope labeled with the study ID and containing the study ID and accompanying 
treatment code. This code will be recorded on the participant’s 2 weeks postnatal questionnaire, and 
the participant will receive their first blister pack of capsules. At every data collection point the 
participant’s study ID will be typed into the data collection tablet and the treatment code will be 
displayed in order to ensure that the participant is receiving the correct treatment throughout the 
study.   
 
8.4 Breaking of the study blind 
 
Breaking blinding during the study 
The master ID list will not be unblinded unless in the unlikely event of an adverse event in the trial. 
Note that the likelihood of any adverse events is extremely unlikely since there is no UL for thiamine 
because there have been no reports of adverse effects of excess thiamine intake (1,2,25). If, however, 
there is a medical emergency and unblinding of a participant is required, our blinding mechanism 
would allow for only one of the two codes per treatment arm to be revealed (as each treatment arm 
will have two codes). We would then document and explain to the funding agency and all ethics 
committees why any premature unblinding occurred.  
 
Breaking the blinding after the study 
Once data collection and cleaning is complete, the database will be locked and unblinded treatment 
codes will be included in the database because a blinded analysis is not possible for estimating dose 
response curves.  
 
Due to the time-consuming nature of coding video-recorded cognitive assessments, data for the 
following assessments will not be included in the database before it is locked and unblinded: primary 
engagement task, secondary engagement task, Visual Paired Comparison Task, and Language 
Preference Task. Note that the research assistants coding the videos for these assessments will 
remain blinded.  
 
8.5 Participant withdrawal 
 
Participants are free to withdraw from the study at any time. The reasons for withdrawal will be 
recorded and reported in a CONSORT-style flow and follow up diagram. Participants who 
discontinue treatment or are withdrawn from the study will not be replaced. Any data collected on 
that participant up to the time of withdrawal will be retained for analysis.  
 
9. Study Procedures 
 
Data and biological samples will be collected at enrolment (between t=0 to 2 weeks postpartum), at 
2, 4, 12, 24, and 52 weeks postnatal, as well as during fortnightly monitoring visits. A pictorial 
summary of data collection can be found in Figure 1.  
 
 

Trial of thiamine supplementation in Cambodia: Protocol 
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9.1 Chart review 
 
Upon enrolment, a primary healthcare worker in the Health Centre (receiving an incentive payment 
from the study) will record data from the dyad’s chart such as any interventions/events at birth, 
maternal age, number of antenatal care visits, number of iron and folic acid tablets consumed, time 
and date of birth, sex of infant, and infant anthropometrics.  
 
9.2 Fortnightly monitoring 
 
Every two weeks between 2-24 weeks, a member of the research team will visit the participant in her 
home to distribute new blister packs and collect used blister packs (compliance measure). At this 
time the field staff will also ask a small number of questions about capsule compliance, selling or 
sharing of salt, and about infant and young child feeding (IYCF) practices (was the infant exclusively 
breastfed over the previous two weeks). The field staff will weigh the household salt container to 
assess salt disappearance, and will provide more salt to the household.  
 
9.3 Salt disappearance 
 
At the initial home visit after randomization, field staff will check that the household has removed 
all salt from the home, and will distribute table salt in specialized study containers. Using calibrated 
scales (sensitive to 1 g), field staff will log the initial weight of the salt container(s). Participants will 
be asked to consume only salt provided by the study, and since Cambodian families eat from a 
common pot, they will instructed for all family meals to be prepared using this salt. At each 
fortnightly monitoring visit, field staff will weigh the salt container(s) to determine household salt 
consumption from salt disappearance. In addition, field staff will ask women to confirm the 
household members eating from the common pot. 
 
The fortnightly salt disappearance study will yield household salt consumption, but does not allow 
us to make strong estimates of intra-household salt consumption, nor the consumption of salt more 
holistically (including from packaged foods, salt-containing condiments). This information will be 
important moving forward in order to calculate dosages for thiamine-fortified salt. Also, gathering 
information not only from lactating women, but also presumably high consumers (husbands) and 
very low consumers at risk of deficiency (children after the window of micronutrient powder 
consumption, so 24-59 months) is also vital for future fortification modelling. Therefore, we will 
collect supplemental information in a randomly selected subset of households (n=100) using 
observed weighed intakes and maternal urinary sodium concentrations (randomly selected from 
larger study, and a second day repeat in 75 households on non-consecutive days). The participant 
(mother), the husband/man in household aged 18-50 years, and a child between 24-59 months 
(husband/man and child will not be applicable in all households) will be enrolled in this sub-study. 
For the observed weighed intake sub-study, field staff will sit in the participants home from dawn 
through dusk, recording, at the individual level, intake for the 1-3 individuals for all table salt and 
salt-containing condiments (e.g. fish and soy sauces) consumed throughout the day. For the mothers 
only, we will assess 24-hour urinary sodium concentrations (25). The mother will be instructed to 
awake, void her urine, record the time, and then collect all urine over the next 24 hours into a 
provided container; the collection will end when the woman will attempt to void urine at the exact 
specified time, 24 hours after initiation of collection. The field staff will visit her home to weigh the 
full container and then take an aliquot for testing (the remaining urine can be discarded). The urine 

Trial of thiamine supplementation in Cambodia: Protocol 
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sample will be placed in the 4°C fridge in Kampong Thom for <1 week before being transported 
the Pasteur Institute in Phnom Penh for storage at -20°C and subsequent batch analysis via 
potentiometry using ion selective electrode on an automated ABX PENTRA C400. We will model 
thiamine fortification of salt using the University of Iowa’s Intake Monitoring, Assessment and 
Planning Program software (http://www.side.stat.iastate.edu/imapp.php). 
 
9.4 Questionnaire 
 
Using an interviewer-administered questionnaire, we will collect demographic and socioeconomic 
information, health information including questions about sleep (adapted from (42)) and postpartum 
depression (43), limited dietary intake data (e.g. perceptions of salt intake, postpartum food taboos 
(44)), and IYCF knowledge and behaviors at 2, 12, 24, and 52 weeks postnatal. 
 
9.5 Anthropometrics 
 
Infants: length, weight, and head circumference; mothers: height and weight. Initial measurements 
will be taken in the Health Centre at delivery, and all other measurements will be collected in 
participant’s homes (2, 12, 24, and 52 weeks postnatal) using calibrated instruments and standard 
protocols as per (45).  
 
9.6 Venous blood samples 
 
Trained, Khmer-speaking phlebotomists will meet mothers and infants at their home or a central 
village location (health centre, or village chief’s home) to collect maternal and infant blood samples 
into EDTA-coated tubes. This is a very low risk procedure; however, participants will be advised 
that the blood collection procedure may cause some discomfort and slight bruising or, very rarely, an 
infection at the site of the needle poke. After the blood draw participants will immediately be given a 
bandage to cover the spot where the blood was taken. Maternal blood samples will be collected at 2 
and 24 weeks postnatal; infant samples will be collected only at 24 weeks postnatal. Time of day and 
time since last meal will be recorded. 
 
9.7 Human milk samples 
 
Human milk samples will be collected using a battery-powered single breast pump (Swing Breast 
pump, Medela) at 2, 4, 12, and 24 weeks postnatal. One full breast expression (single breast) will be 
collected from the breast women self-identify as being more ‘full’ (the breast not most recently 
emptied). Since time of day has little effect on milk thiamin concentrations (46), samples can be 
collected at any time of day, however, time of day, time since last meal, and breast side will be 
recorded.  
 
9.8 Blood and human milk processing and analysis 
 
Biological samples will be collected in the village (woman’s home, or central village location such as 
the village chief’s home), placed on ice, and transported to the field lab in Kampong Thom within 5 
hours of collection. Phlebotomists will collect 9 mL venous blood from mothers and 5 mL from 
infants (in EDTA-coated Vaccutainers). Venous blood samples will be processed as per Figure 2. 
All samples will be stored at -20°C for <1 week before being transported the National Institute for 

Trial of thiamine supplementation in Cambodia: Protocol 
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Public Health Laboratory in Phnom Penh for storage at -80°C. Samples will be batch shipped on dry 
ice after the 24 weeks postnatal data collection is completed. 
 
 
Figure 2: Processing protocol for venous blood and breast milk samples. 
 
Venous blood samples will undergo analysis for both transketolase activity and thiamine 
diphosphate concentrations, as described in 3.3 Biomarkers of thiamine status, by Albert Koulman at the 
NIHR BRC Nutritional Biomarker Laboratory at the University of Cambridge in the United 
Kingdom. At 2 weeks postnatal, maternal whole blood ThDP and erythrocyte transketolase activity 
will be measured. At 24 weeks postnatal, ThDP and transketolase activity will be measured in both 
whole blood and erythrocytes, and infant samples will be assayed for whole blood ThDP and 
erythrocyte transketolase activity. When ThDP is assessed in whole blood, concentrations must be 
normalized to hemoglobin concentrations and/or hematocrit (47). Therefore, hemoglobin 
concentration and hematocrit will be measured on all whole blood samples via a HemoCue 201 
portable hemoglobinometer and capillary hematocrit tubes, respectively. 
 
Evidence from the 2014 Cambodian National Micronutrient survey indicates that eThDP is affected 
by the presence/absence of genetic hemoglobin disorders. With this, it is vital to assess the 
presence/absence of genetic hemoglobin disorders, as this could influence participant’s response to 
thiamine, it may influence transketolase, and it could change thiamine dosage requirements in other 

Trial of thiamine supplementation in Cambodia: Protocol 
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countries. Therefore, at one timepoint only (2 weeks postnatal for maternal, and 24 weeks postnatal 
for infant samples), an aliquot of 1 mL whole blood will undergo hemoglobin capillary 
electrophoresis analysis to identify structural hemoglobin variants (48) at the Pasteur Institute in 
Phnom Penh. Buffy coat samples will be stored in the BioBank in the Department of Applied 
Human Nutrition at Mount Saint Vincent University in Halifax, Nova Scotia, Canada, for potential 
later assessment of genetic hemoglobin disorders. 
 
Biomakers may be influenced by inflammation, which is common in this population (49,50). Plasma 
samples will be sent to Dr. Jurgen Erhardt at the VitMin Lab in Germany for analysis of C-reactive 
protein (CRP) and α-1-acid-glycoprotein (AGP) using an immunosorbent assay (51). Note that this 
assay will also measure retinol binding protein (RBP), ferritin, and soluble transferrin receptor 
(sTfR). Other plasma samples will be stored in the BioBank for potential future use. 
 
At 2, 4, 12, and 24 weeks postnatal, a full human milk expression will be collected (approx. 35 g) and 
approximate milk volume (weight of milk to 1 g) will be recorded. Samples will be mixed, and 2 mL 
aliquots obtained in amber cryovials (see Figure 2). Human milk thiamine concentrations will be 
measured by Drs. Daniela Hampel and Lindsay Allen at the USDA/ARS Western Human Nutrition 
Research Center, University of California, Davis (52).  
 
9.9 Cognitive assessments 
 
We will conduct cognitive assessments at 2, 12, 24, and 52 weeks postnatal; video-recordings of data 
collection will be collected to aid in analysis. We will conduct the Hammersmith Infant Neurological 
Exam (10 minutes), an oculomotor exam (2 minutes), Infant Mullen Scales of Early Learning (10 
minutes at 2 and 12 weeks postnatal, 15 minutes at 24 and 52 weeks postnatal), Caregiver Reported 
Early Development Instruments (CREDI; 15 minutes), a Primary Engagement Task (3 minutes; 2, 
12, and 24 weeks only), a Secondary Engagement Task (2.5 minutes), the Visual Paired Comparison 
Task (7 minutes), and the Language Preference Task (6 minutes), in the women’s home. See Figure 1 
for the timepoints at which these tasks are conducted. At 52 weeks postnatal, we will also 
incorporate the Mother-Infant Free Play Interaction (5 minutes), and the MacArthur-Bates 
Communicative Development Inventory (8 minutes).  
 
The Hammersmith Infant Neurological Exam is a standard tool for providing basic information 
about infants’ neurological status through use of gentle touch and gentle social interaction in order 
to examine infants’ sensory and motor responses; it has previously shown excellent interobserver 
reliability even among less experienced staff (53). An oculomotor exam was included because 
oculomotor disturbances are not uncommon among thiamine deficient adults (54). The Infant 
Mullen Scales of Early Learning is an individually administered, multi-domain measure of early 
development, with scales measuring development in visual reception, fine motor, gross motor, 
receptive language, and expressive language (55). The CREDI is a caregiver-reported measure of 
child development that has been shown to have right validity and reliability in low-resource settings 
(56). The Primary Engagement Task measures individual variation in infant, caregiver, and dyad-
level developmental change in the ability to engage contingently with the partner in direct positive 
mutual engagement interactions. The Secondary Engagement Task measures individual variation in 
caregiver and infant ability to engage mutually in relation to an external object. The Visual Paired 
Comparison task (VPC; 5 minutes) and the Language Preference Task (5 minutes). The VPC probes 
infants’ recognition memory and attention, and has been used to predict subsequent verbal IQ in 

Trial of thiamine supplementation in Cambodia: Protocol 
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middle childhood (57,58), and produce measures of attention predictive of developmental maturity 
and better cognitive function (59,60). The Language Preference Task is designed to measure 
individual variation in infants’ level of interest in the kind of language that caregivers typically direct 
toward infants (aka infant-directed talk or ‘motherese’) relative to a) the kind of language that is 
typically directed toward adults (aka adult-directed talk), and b) sounds that are matched in 
complexity but are non-linguistic (non-linguistic analog). Over a series of trials, infants hear these 
three different kinds of sound samples through an audio speaker (infant-directed talk, adult-directed 
talk, non-linguistic analog), and the duration that they look toward a visual display during each 
sound sample is subsequently measured from the videotaped record collected during the session. 
 
The Mother-Infant Free Play Interaction aims to provide behavioral samples of caregiver-
infant/child interaction dynamics, and have been used extensively by developmental scientists to 
detect and measure typical and atypical developmental trajectories starting at birth (61,62). At 52 
weeks, we will use a free-play task designed to elicit positive affect that we have used successfully in 
Laos with undernourished and healthy caregiver-infant dyads (63). As with our Primary Engagement 
Task at the three earlier study time points, coding of the interactions will target maternal, infant and 
dyadic behaviors, and will be based on Walker and Thompson’s Mother-Infant Play Interaction 
Scale (64).  
 
The MacArthur-Bates Communicative Development Inventory (CDI) is a parent report inventory 
developed to assess language and communication development in children aged 8 to 30 months. We 
propose to use the “short form” of the CDI, which typically takes less than 10 minutes to complete. 
The CDI has been used extensively to study the language development of typical and atypical 
populations (65), and has proven capable of discriminating between groups infants/toddlers who 
differ in terms of birthweight complications and early neurological adversity (66) and suboptimal 
early caregiver environments (67). The CDI has been used successfully in over 20 different 
languages. Presently, a Khmer version does not exist, thus our team will utilize the MacArthur-Bates 
CDI’s “adaptation guidelines” to create a Khmer version. 
 
9.10 Participant remuneration 
 
All participants will receive a mobile phone and mobile phone credits. In addition, modest, study-
appropriate remuneration such as a sarong, canned fish, or soap will be provided at biological 
sample collection/cognitive assessment points (2, 4, 12, 24, and 52 weeks postnatal). 
 
10. Adverse Event Reporting 
 
As described in 2.6 Serious Adverse Events Committee / Data Safety Monitoring Board, there will be no 
Serious Adverse Events Committee or Data Safety Monitoring Board assembled for this study, 
because no adverse events have ever been reported for thiamine. There is no UL for thiamine 
because there has never been an adverse event reported from high thiamine intake (1,2), even 
though over-the-counter supplements in Canada and the United States commonly contain 50 mg 
thiamine or more. With this, we are confident that there is a low risk of serious adverse events in 
this study. There are also no clinical outcomes for which to monitor: the primary outcome of the 
study is a biochemical measure of thiamine status, but results will only be known after study 
completion. Also, there will be no mid-study data available to examine because all biological samples 

Trial of thiamine supplementation in Cambodia: Protocol 
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will be batch analyzed to decrease assessment variability (in addition to logistic considerations for 
batch storing and shipping samples).  
 
However, the study will employ a Cambodian medical doctor from the Kampong Thom 
Operational District to answer medical questions from mothers, and, on an ad hoc basis, to 
investigate adverse events. As described above in 2.6 Serious Adverse Events Committee / Data Safety 
Monitoring Board, we do expect 2 – 4 infant death during the study due to events not related to the 
study.  
 
11. Statistical Analysis 
 
The full statistical analysis plan can be found in Appendix B. The primary analyses will be 
performed on an ‘intention to treat’ basis, where all participants will be analyzed according to their 
allocated treatment group regardless of compliance. A secondary ‘per-protocol’ analysis will be 
performed on the subset of women who consumed ≥80% capsules over the study period. The 
parameters of the Emax dose-response curves will be estimated using non-linear least squares 
models. Separate curves will be fitted for outcomes measured at 4 weeks, 12 weeks, and 24 weeks 
postpartum. The dose where additional maternal intake of thiamine no longer meaningfully increases 
milk thiamine concentration, defined as the dose that achieves 90% of the maximum concentration, 
will be estimated from the fitted curve with standard errors estimated by bootstrapping for 
calculating 95% confidence intervals.  
 
Comparisons between treatment groups will be performed using linear mixed-effects models to 
account for repeated measurements and adjusted for randomization strata and levels at 2 weeks 
postnatal. Interaction tests will be performed to assess whether treatment effects depend on genetic 
hemoglobin disorder. Missing data will be addressed using multiple imputation to create 100 
complete datasets for analysis, with a sensitivity analysis performed on the raw (unimputed) data 
(68). All analyses will follow a pre-specified statistical analysis plan (see Appendix B). 
 
12. Administrative Aspects, and Data Security and Management   
 
12.1 Obtaining informed consent 
 
Field staff will follow up with interested women and screen for eligibility in their homes. If women 
are eligible and wish to participate, the field staff will obtain consent by reading them the consent 
form and obtaining written informed consent via a thumbprint stamp. Signatures are not common 
in Cambodia; instead participants will provide a thumbprint stamp, as is common practice in 
research studies in Cambodia. During this initial meeting, women will be told that they can withdraw 
from the study at any time without penalty. Women will be reminded of the consent process at 2, 4, 
12, 24, and 52 weeks postnatal, before data and biological samples are collected.  
 
If women are not interested or are unwilling to provide consent, the field staff will thank them for 
their time and leave their homes. 
 
As part of the consent process participants will be also be asked if they would provide permission 
for the repurposing of their biological samples after this study to be ‘biobanked’ for use in future 
nutrition-related research (re-consent will not be obtained). Participants will be informed that any 
future use would involve only de-identified data, and would be for nutrition-related research only. 

Trial of thiamine supplementation in Cambodia: Protocol 
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Participants can chose whether or not to provide this consent for future use of samples: the consent 
form is clear that this is secondary use of the biological samples, and is in no way required for 
participation in this study. 
 
12.2 Ethics 
 
We will obtain ethics approvals from the following Research Ethics Boards: 
- National Ethics Committee for Health Research (Cambodia)  
- Mount Saint Vincent University Research Ethics Committee (Canada) 
- University of Oregon Institutional Review Board (USA) 
 
This research will also be registered at clinicaltrials.gov. 
 
12.3 Confidentiality and data security 
 
Participants will be given a unique alpha-numeric study ID code. This unique identifier will not be 
derived from personal identifiers. A key linking the subject code to participant information will be 
kept on a password protected computer in a secure area in the Helen Keller International (HKI) 
office in Phnom Penh, Cambodia. There is no need for co-investigators elsewhere to have 
information linking participant names with their unique identifier.  
 
All electronic data files will be stored on password protected computers and/or secure servers 
accessible only to members of the research team. Archived electronic data files and any hard copies 
of data (consent forms) will be stored in locked filing cabinets in locked research rooms at HKI 
Cambodia.  
 
Once all data is collected, all information will be de-identified. Only the study statistician and 
Principal Investigator will have access to the master list once data collection is complete; otherwise, 
all data will be completely de-identified. 
 
Biological samples will be transported to MSVU on dry ice, and stored in -80°C freezers in a locked 
freezer room. After analysis, all biological samples will be stored for 5 years in -80°C freezers at 
MSVU, then destroyed following biological safety protocols. 
 
All paper copies of consent forms and questionnaires will be retained for at least 5 years after 
publication of results. As per granting agency rules, all data will be made available on a public 
repository, but we will also store data on an external hard-drive, in a locked drawer, in the Principal 
Investigator’s locked office for a minimum of 25 years. After this time, all paper copies of consent 
forms will be shredded, and the hard drive deleted and physically destroyed.  
 
12.4 Modifications to the protocol 
 
This study will be conducted as per the current version of the protocol. Any change to the protocol 
document or related study tools that affects the scientific intent, study design, participant safety, or 
that may affect a participants willingness to continue participation in the study is considered an 
amendment, and therefore will be written and filed as such.   
 

Trial of thiamine supplementation in Cambodia: Protocol 
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12.5 Protocol deviations 
 
Protocol deviations will be documented and reported to the Principal Investigator if data collection 
does not fall within the following windows: 
• chart review: anytime between 0 – 16 days postnatal 
• 2 weeks postnatal: 2 weeks postnatal ± 2 days 
• 4 weeks postnatal: 4 weeks postnatal ± 2 days 
• 12 weeks postnatal: 12 weeks postnatal ± 4 days 
• 24 weeks postnatal: 24 weeks postnatal ± 4 days 
• 52 weeks postnatal: 52 weeks postnatal ± 7 days   
• fortnightly monitoring: ± 2 days 
 
12.6 Trial closure 
 
The Principal Investigator (or designee) may terminate the study prematurely if data become 
available which raise concern about the safety of the study treatment(s) that have potential to cause 
unacceptable risks to subjects. If premature study closure occurs, the Principal Investigator (or 
designee) must contact all participants within two weeks, and written notification must be sent to the 
Ethics Committees.  
 
13. Use of Data, and Dissemination 
 
13.1 Layperson dissemination 
 
We will create various lay outputs from this study that can be used by NGOs and government 
agencies working with families, or can be accessed directly by families. These will include short video 
clips, infographics, and ‘hot tips,’ which we will make available online (website maintained through 
Mount Saint Vincent University; HKI will provide Khmer-translated materials) and in print for 
distribution at local agencies.  
 
We will host a Dissemination Workshop in Phnom Penh open to relevant stakeholders (NGOs, 
government, researchers, media, clinicians, public health, all sectors) to share the main outcomes of 
the study, and officially ‘launch’ resources online. Relevant parties from Myanmar and Laos PDR 
(e.g. Ministry of Health representatives) will be invited to this workshop; we will also engage with 
these stakeholders to explore take-up and/or scaling in these countries.   
 
Updates on the study will be shared at the monthly Cambodian Nutrition Working Group meeting, 
hosted by the National Nutrition Programme, Ministry of Health, and attended by all researchers 
and NGOs engaging in nutrition research and programming in Cambodia. Scaling Up Nutrition 
(SUN) and other relevant groups will also be updated regularly, and we will share dissemination 
materials, and invite these groups to the dissemination workshop. 
 
We will return to each community in Kampong Thom to conduct a village-wide meeting relaying 
study results. 
 

Trial of thiamine supplementation in Cambodia: Protocol 
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13.2 Academic dissemination 
 
Results of this research will be presented at academic nutrition, public health, and psychology 
conferences (i.e. Micronutrient Forum, Canadian Nutrition Society and American Nutrition Society 
Annual Meetings, International Society for Research in Human Milk and Lactation Conference, 
Society for Research in Child Development, etc.), and in peer-reviewed, journals that offer open 
access (e.g. Nutrients, American Journal of Clinical Nutrition, Pediatrics, etc.). 
 
We plan to make data available on a public repository by August 31, 2020.  
 

Trial of thiamine supplementation in Cambodia: Protocol 
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14. References  
 
1.  
Institute of Medicine. Thiamin. In: Dietary Reference Intakes for thiamin, riboflavin, niacin, 
vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington, DC: 
National Academy Press; 1998. p. 58–86.  
2.  
Smithline HA, Donnino M, Greenblatt DJ. Pharmacokinetics of high-dose oral thiamine 
hydrochloride in healthy subjects. BMC Clin Pharmacol. 2012;12:4.  
3.  
National Institute of Statistics, Directorate General for Health, ICF International. Cambodia 
Demographic and Health Survey 2014. Phnom Penh, Cambodia and Rockville, Maryland, 
USA; 2015.  
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Bemeur C, Butterworth RF. Thiamin. In: Ross AC, Calallero B, Cousins RJ, Tucker KL, 
Ziegler TR, editors. Modern Nutrition in Health and Disease. 11th ed. Baltimore, MD: 
Lippincott Williams & Wilkins; 2014. p. 317–24.  
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Khounnorath S, Chamberlain K, Taylor AM, Soukaloun D, Mayxay M, Lee SJ, et al. Clinically 
unapparent infantile thiamin deficiency in Vientiane, Laos. PLoS Negl Trop Dis. 
2011;5(2):e969.  
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Coats D, Shelton-Dodge K, Ou K, Khun V, Seab S, Sok K, et al. Thiamine deficiency in 
Cambodian infants with and without beriberi. J Pediatr. 2012;161(5):843–7.  
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Porter SG, Coats D, Fischer PR, Ou K, Frank EL, Sreang P, et al. Thiamine deficiency and 
cardiac dysfunction in Cambodian infants. J Pediatr. 2014;164(6):1456–61.  
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Keating EM, Nget P, Kea S, Kuong S, Daly L, Phearom S, et al. Thiamine deficiency in 
tachypnoeic Cambodian infants. Paediatr Int Child Health. 2014;35(4):312–8.  
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Whitfield KC, Karakochuk CD, Liu Y, McCann A, Talukder A, Kroeun H, et al. Poor 
thiamin and riboflavin status is common among women of childbearing age in rural and 
urban Cambodia. J Nutr. 2015;145(3):628–33.  
10.  
Whitfield KC, Karakochuk CD, Kroeun H, Hampel D, Sokhoing L, Chan BB, et al. Perinatal 
consumption of thiamine-fortified fish sauce in rural Cambodia: a randomized clinical 
efficacy trial. JAMA Pediatr. 2016;170(10):e162065.  
11.  
Whitfield KC, Karakochuk CD, Kroeun H, Sokhoing L, Chan BB, Borath M, et al. 
Household consumption of thiamin-fortified fish sauce increases erythrocyte thiamin 
concentrations among rural Cambodian women and their children younger than 5 years of 
age: a randomized controlled efficacy trial. J Pediatr. 2017;181:242–7.  
12.  
Barennes H, Sengkhamyong K, René JP, Phimmasane M. Beriberi (thiamine deficiency) and 
high infant mortality in northern Laos. PLoS Negl Trop Dis. 2015;9(3):e0003581.  
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Mayxay M, Taylor AM, Khanthavong M, Keola S, Pongvongsa T, Phompida S, et al. Thiamin 
deficiency and uncomplicated falciparum malaria in Laos. Trop Med Int Heal. 
2007;12(3):363–9.  
14.  
Soukaloun D, Lee SJ, Chamberlain K, Taylor AM, Mayxay M, Sisouk K, et al. Erythrocyte 
transketolase activity, markers of cardiac dysfunction and the diagnosis of infantile beriberi. 
PLoS Negl Trop Dis. 2011;5(2):e971.  
15.  
Luxemburger C, White NJ, ter Kuile F, Singh HM, Allier-Frachon I, Ohn M, et al. Beri-beri: 
the major cause of infant mortality in Karen refugees. Trans R Soc Trop Med Hyg. 
2003;97(2):251–5.  
16.  
McGready R, Simpson JA, Cho T, Dubowitz L, Changbumrung S, Böhm V, et al. 
Postpartum thiamine deficiency in a Karen displaced population. Am J Clin Nutr. 
2001;74(6):808–13.  
17.  
FAO Statistics Division. Food Balance Sheets: Cambodia (2011) [Internet]. 2014. Available 
from: http://faostat.fao.org/site/368/DesktopDefault.aspx?PageID=368#ancor 

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Page 26 of 29 
18.  
Allen LH. B vitamins in breast milk: relative importance of maternal status and intake, and 
effects on infant status and function. Adv Nutr. 2012;3(3):362–9.  
19.  
Fattal-Valevski A, Kesler A, Sela B-A, Nitzan-Kaluski D, Rotstein M, Mesterman R, et al. 
Outbreak of life-threatening thiamine deficiency in infants in Israel caused by a defective soy-
based formula. Pediatrics. 2005 Feb;115:233–8.  
20.  
Harel Y, Zuk L, Guindy M, Nakar O, Lotan D, Fattal-Valevski A. The effect of subclinical 
infantile thiamine deficiency on motor function in preschool children. Matern Child Nutr. 
2017;e12397.  
21.  
Fattal-Valevski A, Azouri-Fattal I, Greenstein YJ, Guindy M, Blau A, Zelnik N. Delayed 
language development due to infantile thiamine deficiency. Dev Med Child Neurol. 
2009;51(8):629–34.  
22.  
Fattal I, Friedmann N, Fattal-Valevski A. The crucial role of thiamine in the development of 
syntax and lexical retrieval: a study of infantile thiamine deficiency. Brain. 2011;134(6):1720–
39.  
23.  
Prado EL, Dewey KG. Nutrition and brain development in early life. Nutr Rev. 2014 Apr 
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nutrient requirements. Washington, DC, USA: Institute of Medicine of the National 
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25.  
Gibson RS. Principles of Nutritional Assessment. 2nd ed. New York, NY, USA: Oxford 
University Press; 2005. 908 p.  
26.  
Hampel D, Allen LH. Analyzing B-vitamins in human milk: methodological approaches. Crit 
Rev Food Sci Nutr. 2016;56:494–511.  
27.  
Lu J, Frank EL. Rapid HPLC measurement of thiamine and its phosphate esters in whole 
blood. Clin Chem. 2008;54(5):901–6.  
28.  
Whitfield KC, Smith G, Chamnan C, Karakochuk CD, Sophonneary P, Kuong K, et al. High 
prevalence of thiamine (vitamin B1) deficiency in early childhood among a nationally 
representative sample of Cambodian women of childbearing age and their children. PLoS 
Negl Trop Dis [Internet]. 2017;11(9):e0005814. Available from: https://doi.org/10.1371/ 
journal.pntd.0005814 
29.  
Butterworth RF. Maternal thiamine deficiency: still a problem in some world communities. 
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30.  
Allen LH, de Benoist B, Dary O, Hurrell R. Guidelines on food fortification with 
micronutrients. Geneva, Switzerland: World Health Organization and Food and Agriculture 
Organization of the United Nations; 2006. 376 p.  
31.  
Dwyer JT, Wiemer KL, Dary O, Keen CL, King JC, Miller KB, et al. Fortification and health: 
challenges and opportunities. Adv Nutr. 2015;6:124–31.  
32.  
Smith G. Micronutrient fortification of food: issues for Asia. J Nutr Sci Vitaminol (Tokyo). 
2015;61(Suppl):S183-5.  
33.  
Hurrell R. The Mineral Fortification of Foods. Surrey, UK: Leatherhead Publishing; 1999. 
313 p.  
34.  
Andersson M, Takkouche B, Egli I, Allen HE, de Benoist B. Current global iodine status and 
progress over the last decade towards the elimination of iodine deficiency. Bull World Health 
Organ. 2005;83(7):518–25.  
35.  
De Wals P, Tairou F, Van Allen MI, Uh S-H, Lowry RB, Sibbald B, et al. Reduction in 
neural-tube defects after folic acid fortification in Canada. N Engl J Med. 2007;357(2):135–
42.  
36.  
Sacco J. Food fortification policy in Canada. In: Preedy VR, Srirajaskanthan R, Patel VB, 

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editors. Handbook of food fortification and health: from concepts to public health 
applications. New York, USA: Springer Science+Business Media; 2013. p. 371–84.  
37.  
Nilles EJ, Manaia A, Ruaia B, Huppatz C, Ward C, George P, et al. Re-emergence of thiamine 
deficiency disease in the Pacific islands (2014 – 15): a case-control study. PLoS One. 
2018;13(6):e0198590.  
38.  
Adamolekun B, Hiffler L. A diagnosis and treatment gap for thiamine deficiency disorders in 
sub-Saharan Africa? Ann N Y Acad Sci. 2017;1408(1):15–9.  
39.  
United Nations Children’s Fund (UNICEF). Improving child nutrition: the achievable 
imperative for global progress. New York, USA; 2013. 132 p.  
40.  
Laillou A, Mam B, Oeurn S, Chea C. Iodized salt in Cambodia: trends from 2008 to 2014. 
Nutrients. 2015;7(6):4189–98.  
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Switzerland; 2012.  
42.  
Keller MA, Goldbery WA. Co-sleeping: help or hinderance for young children’s 
independence? Infant Child Dev. 2004;13:369–88.  
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Gjerdingen D, Crow S, McGovern P, Miner M, Center B. Postpartum depression screening at 
well-child visits: validity of a 2-question screen and the PHQ-9. Ann Fam Med. 2009;7:63–70.  
44.  
Wallace LJ, Summerlee AJS, Dewey CE, Hak C, Hall A, Charles C V. Women’s nutrient 
intakes and food-related knowledge in rural Kandal province, Cambodia. Asia Pac J Clin 
Nutr. 2014;23(2):263–71.  
45.  
Cogill B. Anthropometric Indicators Measurement Guide. Washington, DC; 2003.  
46.  
Hampel D, Shahab-Ferdows S, Islam MM, Peerson JM, Allen LH. Vitamin concentrations in 
human milk vary with time within feed, circadian rhythym, and single-dose supplementation. 
J Nutr. 2017;147(4):603–11.  
47.  
Floridi A, Pupita A, Palmerini C, Fini C, Alberti Fidanza A. Thiamine pyrophosphate 
determination in whole blood and erythrocytes by high performance liquid chromatography. J 
Int Vitaminol Nutr. 1983;54:165–171.  
48.  
Hartwell SK, Srisawang B, Kongtawelert P, Christian GD, Grudpan K. Review on screening 
and analysis techniques for hemoglobin variants and thalassemia. Talanta. 2005;65(5):1149–
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49.  
Makurat J, Friedrich H, Kuong K, Wieringa FT, Chamnan C, Krawinkel MB. Nutritional and 
micronutrient status of female workers in a garment factory in Cambodia. Nutrients. 
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Yong T-S, Chai J-Y, Sohn W-M, Eom KS, Jeoung H-G, Hoang E-H, et al. Prevalence of 
intestinal helminths among inhabitants of Cambodia (2006-2011). Korean J Parasitol. 
2014;52(6):661–6.  
51.  
Erhardt JG, Estes JE, Pfeiffer CM, Biesalski HK, Craft NE. Combined measurement of 
ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein by an 
inexpensive, sensitive, and simple sandwich enzyme-linked immunosorbent assay technique. J 
Nutr. 2004;134(11):3127–32.  
52.  
Hampel D, York ER, Allen LH. Ultra-performance liquid chromatography tandem mass-
spectrometry (UPLC-MS/MS) for the rapid, simultaneous analysis of thiamin, riboflavin, 
flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk. J Chromatogr B. 
2012;903:7–13.  
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Maitre NL, Chorna O, Romeo DM, Guzzetta A. Implementation of the Hammersmith 
Infant Neurological Examination in a high-risk infant follow-up program. Pediatr Neurol. 
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Service Inc.; 1995.  
56.  
McCoy DC, Sudfeld CR, Bellinger DC, Muhihi A, Ashery G, Weary TE, et al. Development 
and validation of an early childhood development scale for use in low-resourced settings. 
Popul Health Metr. 2017;15(3).  
57.  
McCall RB. What process mediates predictions of childhood IQ from infant habituation and 
recognition memory? Speculations on the roles of inhibition and rate of information 
processing. Intelligence. 1994;18(2):107–25.  
58.  
Sigman M, Cohen SE, Beckwith L. Why does infant attention predict adolescent intelligence? 
Infant Behav Dev. 1997;20(2):133–40.  
59.  
Rose SA, Feldman JF, Jankowski JJ. Dimensions of cognition in infancy. Intelligence. 
2004;32:245–62.  
60.  
Rose SA, Feldman JF, Jankowski JJ, Rossem R Van. A cognitive cascade in infancy: Pathways 
from prematurity to later mental development. Intelligence. 2008;36(4):367–78.  
61.  
Hall RAS, Hoffenkamp HN, Tooten A, Braeken J, Vingerhoets AJJM, van Bakel HJA. The 
quality of parent–infant interaction in the first 2 years after full-term and preterm birth. 
Parent Sci Pract. 2015;15(4):247–68.  
62.  
Bernier A, Salkins SD, Bell MA. Longitudinal associations between the quality of mother-
infant interactions and brain development across infancy. Child Dev. 2016;87(4):1159–74.  
63.  
Measelle JR, Mobasser A, Fong M, Soulalay CS, Nijssen-Jordan C. Developmental 
neuroscience and stunting: a strong case for action in the first 1000 days. Ann Glob Heal. 
2016;82(3):514.  
64.  
Walker LO, Thompson ET. Analysis of current assessment strategies in the health care of 
young children and childbearing families. In: Humenick SS, editor. Mother–infant play 
interaction scale. East Norwalk, CT, USA: Appleton-Century-Crofts; 1982. p. 191–201.  
65.  
Heilmann J, Ellis Weismer S, Evans J, Hollar C. Utility of the MacArthur-Bates 
communicative development inventory in identifying language abilities of late-talking and 
typically developing toddlers. Am J Speech-Language Pathol. 2005;14(1):40–51.  
66.  
Chilosi AM, Cipriani P, Bertuccelli B, Pfanner L, Cioni G. Early cognitive and 
communication development in children with focal brain lesions. J Child Neurol. 
2001;16(5):309–16.  
67.  
Lyytinen P, Eklund K, Lyytinen H. The play and language behavior of mothers with and 
without dyslexia and its association to their toddlers’ language development. J Learn Disabil. 
2003;36(1):74–86.  
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Graham JW, Olchowski AE, Gilreath TD. How many imputations are really needed? Some 
practical clarifications of multiple imputation theory. Prev Sci. 2007;8(3):206–13.  
 
 
  

Trial of thiamine supplementation in Cambodia: Protocol 
Page 29 of 29 
Appendices 
 
Appendix A: Thiamine hydrochloride safety data sheet. 
 
Appendix B: Statistical Analysis Plan for Trial of thiamine supplementation in Cambodia. 
 
 

SAFETY DATA SHEET
SECTION 1: PRODUCT IDENTIFICATION
THIAMINE HYDROCHLORIDE, USP (Vitamin B1)
PRODUCT NAME
1963
PRODUCT CODE
MEDISCA Inc.
SUPPLIER
Tel.: 1.800.932.1039 | Fax.: 1.855.850.5855
661 Route 3, Unit C, Plattsburgh, NY, 12901
3955 W. Mesa Vista Ave., Unit A-10, Las Vegas, NV, 89118
6641 N. Belt Line Road, Suite 130, Irving, TX, 75063
MEDISCA Pharmaceutique Inc.
Tel.: 1.800.665.6334 | Fax.: 514.338.1693
4509 Rue Dobrin, St. Laurent, QC, H4R 2L8
21300 Gordon Way, Unit 153/158, Richmond, BC V6W 1M2
MEDISCA Australia PTY LTD
Tel.: 1.300.786.392 | Fax.: 61.2.9700.9047
Unit 7, Heritage Business Park
5-9 Ricketty Street, Mascot, NSW 2020
CHEMTREC Day or Night Within USA and Canada: 1-800-424-9300
NSW Poisons Information Centre: 131 126
EMERGENCY PHONE
Supplement
USES
SECTION 2: HAZARDS IDENTIFICATION
Based on available data, the classification criteria are not met.
GHS CLASSIFICATION
Not Applicable
PICTOGRAM
Not Applicable
SIGNAL WORD
Not Applicable
HAZARD STATEMENT(S)
 Not Applicable.
AUSTRALIA-ONLY HAZARDS
Not Applicable
PRECAUTIONARY STATEMENT(S)
Prevention
Not Applicable
Response
Not Applicable
Storage
Disposal
 0
 0
HMIS CLASSIFICATION
Health Hazard
Flammability
 0
 B
Reactivity
Personal Protection
SECTION 3: COMPOSITION/INFORMATION ON INGREDIENTS
Thiazolium, 3-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-5-(2-hydroxyethyl)-4-methyl-, chloride, 
monohydrochloride
CHEMICAL NAME
Not applicable
BOTANICAL NAME
Vitamin B1 hydrochloride
SYNONYM
C₁₂H₁₇ClN₄OS.HCl
CHEMICAL FORMULA
67-03-8
CAS NUMBER
THIAMINE HYDROCHLORIDE, USP (Vitamin B1)
Page 1 of 6
Last Revision: 12/2015

SAFETY DATA SHEET
Not applicable
ALTERNATE CAS NUMBER
MOLECULAR WEIGHT
337.3
% BY WEIGHT
CAS NUMBER
CHEMICAL NAME
100
67-03-8
THIAMINE HYDROCHLORIDE
COMPOSITION
There are no additional ingredients present which, within the current knowledge of the supplier and in the 
concentrations applicable, are classified as health hazards and hence require reporting in this section.
SECTION 4: FIRST-AID MEASURES
Flush with copious amounts of water for 15 minutes, separating eyelids with fingers. If irritation persists seek 
medical aid.
IN CASE OF EYE CONTACT
Wash with soap & water for 15 minutes. If irritation persists seek medical aid.
IN CASE OF SKIN CONTACT
Call a physician. Wash out mouth with water. Do not induce vomiting without medical advice.
IF SWALLOWED
Remove to fresh air.  If not breathing, give artificial respiration.  If breathing is difficult, give oxygen. Call a 
physician
IF INHALED
Not expected to present a significant hazard under anticipated conditions of normal use.
SYMPTOMS AND EFFECTS
SECTION 5: FIREFIGHTING MEASURES
Not applicable
SPECIFIC HAZARDS ARISING FROM 
THE CHEMICAL
May be combustible at high temperature
FLAMMABLE PROPERTIES
Under fire conditions, hazardous fumes will be present.
HAZARDOUS COMBUSTION PRODUCTS
Small fire: dry chemical, CO₂ or water spray. Large fire: dry chemical, CO₂, alcohol resistant foam or water 
spray. Do not get water inside containers.
EXTINGUISHING MEDIA
Wear self-contained breathing apparatus and protective clothing to prevent contact with skin and eyes.
PROTECTIVE EQUIPMENT AND 
PRECAUTIONS FOR FIREFIGHTERS
SECTION 6: ACCIDENTAL RELEASE MEASURES
Wear respiratory protection. Avoid dust formation. Avoid breathing vapours, mist or gas. Ensure adequate 
ventilation. Evacuate personnel to safe areas. Avoid breathing dust.
PERSONAL PRECAUTIONS
On land, sweep or shovel into suitable containers.  Minimize generation of dust.
METHODS & MATERIAL FOR 
CONTAINMENT
Do not touch damaged containers or spilled material unless wearing appropriate protective clothing. Wear 
respirator, chemical safety goggles, rubber boots and heavy rubber gloves. Stop leak if you can do it without 
risk. Prevent entry into waterways, sewers, basements or confined areas. Shut off all sources of ignition. 
Evacuate the area.  If necessary, employ water fog to disperse the vapors. Absorb the matter with compatible 
vermiculite or other absorbing material. Place in a suitable container and retain for disposal. Ventilate and clean 
the affected area. Do not flush into sewerage system or to drains.
CLEANUP PROCEDURE
SECTION 7: HANDLING AND STORAGE
THIAMINE HYDROCHLORIDE, USP (Vitamin B1)
Page 2 of 6
Last Revision: 12/2015

SAFETY DATA SHEET
Do not inhale. Avoid contact with eyes, skin and clothing. Avoid prolonged or repeated exposure. Wash 
thoroughly after handling.
PRECAUTIONS FOR SAFE HANDLING
Store in original container, tightly sealed, protected from direct sunlight, in a dry, room temperature and 
well-ventilated area, away from incompatible materials. Store in accordance with local regulations. Eliminate all 
ignition sources. Separate from oxidizing materials. Containers that have been opened must be carefully 
resealed and kept upright to prevent leakage. Do not store in unlabelled containers. Use appropriate 
containment to avoid environmental contamination. Use non-metalic containers. 
Preserve in tight, light-resistant containers.
STORAGE CONDITIONS
SECTION 8: EXPOSURE CONTROLS/ PERSONAL PROTECTION
Chemical Name: THIAMINE HYDROCHLORIDE    CAS #: 67-03-8
IDLH
REL
STEL
Ceiling
TWA
Remarks
NIOSH
N/L
N/L
N/L
N/L
N/L
-
AIHA WEEL
N/L
N/L
N/L
-
-
-
Safe Work Australia HSIS
N/L
N/L
N/L
-
-
-
HSE
N/L
N/L
N/L
-
-
-
OSHA PEL
N/L
N/L
-
-
-
-
ACGIH TLV
N/L
N/L
N/L
-
-
-
N/L = Not Listed
Consult local authorities for provincial or state exposure limits. Particulates not otherwise regulated, respirable 
fraction: 5 mg/m3.
EXPOSURE GUIDELINES
Eyes: Wear appropriate protective eyeglasses or chemical safety goggles as described by WHMIS or OSHA's 
eye and face protection regulations in 29 CFR 1910.133 or European Standard EN166. Skin: Wear appropriate 
gloves to prevent skin exposure. Clothing: Wear appropriate protective clothing to minimize contact with skin. 
Respirators: Follow WHMIS or OSHA respirator regulations found in 29 CFR 1910.134 or European Standard 
EN 149. Use a NIOSH/MSHA or European Standard EN 149 approved respirator if exposure limits are 
exceeded or if irritation or other symptoms are experienced.
PERSONAL PROTECTIVE EQUIPMENT
Adequate mechanical ventilation. Fumehood, eye wash station, and safety shower.
SPECIFIC ENGINEERING CONTROLS
SECTION 9: PHYSICAL AND CHEMICAL PROPERTIES
Solid
PHYSICAL STATE
White to off-white crystals or crystalline powder, usually having a slight, characteristic odor. Is hygroscopic.
DESCRIPTION
Freely soluble in water; soluble in glycerin; slightly soluble in alcohol; insoluble in ether and in benzene.
SOLUBILITY
Characteristic nut-like odor.
ODOR
May be combustible at high temperature
FLAMMABILITY
2.7 - 3.4 (1%)
248 °C, 478.4 °F 
(decomposes)
Not available
ODOR THRESHOLD
pH
MELTING POINT
Not available
Not available
Not available
BOILING POINT
FREEZING POINT
FLASH POINT
1.4 (30 °C)
Not available
Not available
EVAPORATION RATE
EXPLOSIVE LIMIT
SPECIFIC GRAVITY
THIAMINE HYDROCHLORIDE, USP (Vitamin B1)
Page 3 of 6
Last Revision: 12/2015

SAFETY DATA SHEET
Not available
Not available
Not available
UPPER FLAMMABLE/ 
EXPLOSIVE LIMIT(S)
LOWER FLAMMABLE/ 
EXPLOSIVE LIMIT(S)
VAPOR PRESSURE
Not available
Not available
-3.930
VAPOR DENSITY 
(AIR = 1)
RELATIVE DENSITY 
(WATER = 1)
log P 
(OCTANOL-WATER)
Not available
Not available
Not available
DECOMPOSITION 
TEMPERATURE
VISCOSITY
AUTO-IGNITION 
TEMPERATURE
SECTION 10: STABILITY AND REACTIVITY
Not established
REACTIVITY
Stable under recommended storage conditions
STABILITY
Alkalis. Oxidizing agents. Reducing agents.
MATERIALS TO AVOID
Toxic fumes of carbon monoxide, carbon dioxide, nitrogen oxides and other gases may occur
HAZARDOUS DECOMPOSITION 
PRODUCTS
Will not occur
HAZARDOUS POLYMERIZATION
Not established
POSSIBLITY OF HAZARDOUS 
REACTION
Moisture, sunlight and extreme temperatures
CONDITIONS TO AVOID
SECTION 11: TOXICOLOGICAL INFORMATION
Oral: Rat: LD50: (mg/kg): 3710
Dermal: Rabbit: LD50: (mg/kg): Not available
Inhalation: Rat: LD50: (mg/L/4hr): Not available
ACUTE TOXICITY
Due to lack of data the classification is not possible.
SKIN CORROSION/IRRITATION
Due to lack of data the classification is not possible.
SERIOUS EYE DAMAGE/EYE 
IRRITATION
Due to lack of data the classification is not possible.
RESPIRATORY OR SKIN 
SENSITIZATION
Due to lack of data the classification is not possible.
Data from germ cell mutagenicity tests were not found.
GERM CELL MUTAGENICITY
THIAMINE HYDROCHLORIDE is not listed.
CARCINOGENICITY
OSHA
THIAMINE HYDROCHLORIDE is not listed.
NTP
THIAMINE HYDROCHLORIDE is not evaluated.
IARC
This product does not contain any chemicals known to the State of California to cause 
cancer, birth defects, or any other reproductive harm.
California 
Proposition 65
ADDITIONAL CARCINOGENICITY 
INFORMATION
Not available
Due to lack of data the classification is not possible.
REPRODUCTIVE TOXICITY
Due to lack of data the classification is not possible.
SPECIFIC TARGET ORGAN TOXICITY - 
SINGLE EXPOSURE
Due to lack of data the classification is not possible.
SPECIFIC TARGET ORGAN TOXICITY - 
REPEATED EXPOSURE
THIAMINE HYDROCHLORIDE, USP (Vitamin B1)
Page 4 of 6
Last Revision: 12/2015

SAFETY DATA SHEET
Based on available data, the classification criteria are not met.
ASPIRATION HAZARDS
Not expected to present a significant hazard under anticipated conditions of normal use. Adverse effects with 
thiamine are rare, but hypersensitivity reactions have occurred, mainly after parenteral doses. These reactions 
have ranged in severity from very mild to, very rarely, fatal anaphylactic shock.
SIGNS AND SYMPTOMS OF 
EXPOSURE
May be harmful if inhaled. May cause respiratory tract irritation.
POTENTIAL HEALTH EFFECTS
Inhalation
May be harmful if swallowed.
Ingestion
May be harmful if absorbed through skin. May cause skin irritation.
Skin
May cause eye irritation.
Eyes
SECTION 12: ECOLOGICAL INFORMATION
EC50: 48 Hr: Daphnia magna: (mg/L): Not available
LC50: 96 Hr: Fish: (mg/L): Not available
IC50: 72 Hr: Algae: (mg/L): Not available
TOXICITY
Not available
PERSISTENCE AND DEGRADABILITY
Not available
BIOACCUMULATIVE POTENTIAL
Freely soluble in water.
MOBILITY IN SOIL
Not available
OTHER ADVERSE EFFECTS
This product is not intended to be released into the environment
SECTION 13: DISPOSAL CONSIDERATIONS
Dispose of in accordance with federal / local laws and regulations. Avoid release into the environment.
WASTE DISPOSAL
SECTION 14: TRANSPORT INFORMATION
UNITED STATES & CANADA
Not dangerous good
UN PROPER SHIPPING NAME
Not applicable
UN NUMBER
Not applicable
CLASS
Not applicable
PACKING GROUP
AUSTRALIA
Not dangerous good
UN PROPER SHIPPING NAME
Not applicable
UN NUMBER
Not applicable
CLASS
Not applicable
PACKING GROUP
Not applicable
HAZCHEM
Not available
ENVIRONMENTAL HAZARDS
Not applicable
SPECIAL SHIPPING INFORMATION
THIAMINE HYDROCHLORIDE, USP (Vitamin B1)
Page 5 of 6
Last Revision: 12/2015

SAFETY DATA SHEET
SECTION 15: REGULATORY INFORMATION
CERCLA
40 CFR 
Part 302.4
SARA (Title III)
40 CFR
Part 372.65
California
Prop 65
Massachusetts
New Jersey
Right-to-know
Pennsylvania
EPA 40 CFR Part 355
Appendix B
Appendix A
Chemical Name 
& CAS
N/L
N/L
N/L
N/L
N/L
N/L
N/L
N/L
THIAMINE HYDROCHLORIDE 
67-03-8
N/L = Not Listed; X = Listed
AUSTRALIAN REGULATIONS
Poisons and Therapeutic 
Goods Regulation
Therapeutic Goods 
Act
Chemical Name
& CAS
Code of Practices - 
Illicit Drug 
Precursors
THIAMINE HYDROCHLORIDE 
67-03-8
N/L
N/L
N/L
SECTION 16: OTHER INFORMATION
Available upon request
REFERENCES
CAS – Chemical Abstract Service; GHS – Global Harmonized System; OSHA PEL – Occupational Safety & 
Health Administration Permissible Exposure Limits; TWA – Time Weighted Average; HSIS – Hazardous 
Substances Information System; STEL – Short Term Exposure Limit; AIHA WEEL – American Industrial 
Hygiene Association Workplace Environment Exposure Levels; LD50 – Lethal Dose, 50%; IARC – International 
Agency for Research on Cancer; NTP – National Toxicology Program; WHMIS – Workplace Hazardous 
Materials Information System; SARA – Superfund Amendments and Reauthorization Act; EPA – Environmental 
Protection Agency; CERCLA – Comprehensive Environmental Response, Compensation, and Liability Act; 
HMIS – Hazardous Materials Information System; NIOSH – National Institute for Occupational Safety and 
Health; MSHA - Mine Safety and Health Administration; ACGIH - American Conference of Governmental 
Industrial Hygienists; IDHL - Immediately Dangerous to Health or Life; TLV – Threshold Limit Value; HSE – 
Health and Safety Executive; REL - Recommended Exposure Limit
ABBREVIATIONS AND ACRONYMS
12/2015
LAST REVISION
09/2013
SUPERSEDES
This document was created in accordance with OSHA, Safe Work Australia and WHMIS regulations. The above 
information is believed to be correct but does not purport to be all inclusive and shall be used only as a guide. 
The information in this document is based on the present state of our knowledge and is applicable to the 
product with regard to appropriate safety precautions. It does not represent any guarantee of the properties of 
the product. MEDISCA® shall not be held liable for any damage resulting from handling or from contact with the 
above product. Recipients of the product must take responsibility for observing existing laws and regulations.
DISCLAIMER
THIAMINE HYDROCHLORIDE, USP (Vitamin B1)
Page 6 of 6
Last Revision: 12/2015

 
 
Trial of Thiamine Supplementation in Cambodia 
STATISTICAL ANALYSIS PLAN 
 
 
SAP Authors: 
Shalem Leemaqz (Statistician) 
Lisa Yelland (Senior Statistician) 
SAP Version: 
1.7 
SAP Date: 
14 August 2019 
 
 
Approved by: 
 
Signature 
Date 
Kyly Whitfield (Principal Investigator) 
………………………….. …………………… 
Hou Kroeun (Co-Investigator) 
………………………….. …………………… 
 
 
August 14, 2019
14 August 2019

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TABLE OF CONTENTS 
 
1 
PREFACE ........................................................................................................................... 3 
2 
STUDY OBJECTIVES ...................................................................................................... 3 
2.1 
Primary Objective ....................................................................................................... 3 
2.2 
Secondary Objectives .................................................................................................. 3 
3 
STUDY METHODS .......................................................................................................... 4 
3.1 
Overall Study Design .................................................................................................. 4 
3.2 
Selection of study population ...................................................................................... 4 
3.2.1 
Inclusion criteria .................................................................................................. 4 
3.2.2 
Exclusion criteria ................................................................................................. 5 
3.3 
Treatment groups ......................................................................................................... 5 
3.4 
Method of treatment group assignment and randomisation ........................................ 5 
3.5 
Blinding ....................................................................................................................... 6 
3.6 
Sample size .................................................................................................................. 6 
4 
SEQUENCE OF PLANNED ANALYSIS ......................................................................... 6 
4.1 
Interim analysis ........................................................................................................... 6 
4.2 
Final analysis and reporting ........................................................................................ 6 
5 
GENERAL ISSUES FOR STATISTICAL ANALYSIS ................................................... 7 
5.1 
Analysis software ........................................................................................................ 7 
5.2 
Analysis approach ....................................................................................................... 7 
5.3 
Methods for withdrawals, missing data and outliers ................................................... 7 
5.4 
Protocol violations and deviations .............................................................................. 7 
5.5 
Data transformations ................................................................................................... 7 
5.6 
Covariates for adjustment ............................................................................................ 7 
5.7 
Planned subgroup analyses .......................................................................................... 8 
5.8 
Multiple comparisons and multiplicity ....................................................................... 8 
6 
DESCRIPTIVE STATISTICS ........................................................................................... 9 
6.1 
Participant flow ........................................................................................................... 9 
6.2 
Baseline characteristics (2 weeks postnatal) ............................................................. 10 
6.3 
Measurement of treatment compliance ..................................................................... 10 
6.4 
Missing data .............................................................................................................. 10 
6.5 
Randomisation errors ................................................................................................ 10 
7 
STATISTICAL ANALYSES ........................................................................................... 10 
7.1 
Emax curve ................................................................................................................ 11 
7.2 
Primary outcome variable ......................................................................................... 12 
7.2.1 
Human milk total thiamine concentration (24 weeks) ....................................... 12 
7.3 
Secondary outcome variables .................................................................................... 12 
7.3.1 
Infant thiamine diphosphate concentration (24 weeks) ..................................... 12 
7.3.2 
Human milk total thiamine concentration (4 weeks and 12 weeks) ................... 12 
7.3.3 
Infant transketolase activity coefficient (24 weeks) ........................................... 13 

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7.3.4 
Maternal thiamine diphosphate concentration (24 weeks) ................................ 13 
7.3.5 
Maternal transketolase activity coefficient (24 weeks) ...................................... 13 
7.3.6 
Household salt intake ......................................................................................... 14 
7.3.7 
Individual salt intake .......................................................................................... 14 
7.3.8 
Sodium intake ..................................................................................................... 14 
7.3.9 
Mullen (composite and 5 subscales) .................................................................. 14 
7.3.10 
VPC novelty score and attention and processing speed subscales .................... 15 
7.3.11 
Language Preference Task score ....................................................................... 15 
7.3.12 
Oculomotor scores ............................................................................................. 15 
8 
REFERENCES ................................................................................................................. 15 
 
 
 

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1 
PREFACE 
This statistical analysis plan (SAP) describes the planned analyses for the trial: 
Trial of thiamine supplementation in Cambodia. 
The following documents were reviewed in preparation of this SAP: 
• Trial of thiamine supplementation in Cambodia: Protocol version 2.1 (dated 24 July 
2018) 
• Questionnaires and consent forms (dated 6 April 2018 and 19 July 2018) 
 
2 
STUDY OBJECTIVES 
2.1 
Primary Objective 
The primary objective is to estimate the dose of maternal intake of thiamine where the dose-
response curve reaches 90% of the maximum average human milk total thiamine 
concentration at 24 weeks postpartum. 
2.2 
Secondary Objectives 
1. To estimate the dose on the dose-response curve where 90% of the maximum average 
is reached in the following outcomes: 
a. Infant thiamine diphosphate concentrations (ThDP) at 24 weeks postnatal 
b. Human milk total thiamine concentration at 4 and 12 weeks postpartum 
c. Infant transketolase activity coefficient (TKac) at 24 weeks postnatal 
d. Maternal ThDP at 24 weeks postpartum 
e. Maternal TKac at 24 weeks postpartum 
and assess whether this depends on the presence/absence of genetic hemoglobin 
disorder for the following outcomes: 
a. Infant ThDP at 24 weeks postnatal 
b. Infant TKac at 24 weeks postnatal 
c. Maternal ThDP at 24 weeks postpartum 
d. Maternal TKac at 24 weeks postpartum 
2. To test for differences between the 4 randomised groups on: 
a. Human milk total thiamine at 4, 12, and 24 weeks postpartum 
b. Maternal ThDP at 24 weeks postpartum 
c. Maternal TKac at 24 weeks postpartum 
and assess whether this depends on the presence/absence of genetic hemoglobin 
disorder for the following outcomes: 

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a. Maternal ThDP at 24 weeks postpartum 
b. Maternal TKac at 24 weeks postpartum 
3. To estimate the usual household salt intake from mean fortnightly salt disappearance 
(weight lost, in g). 
4. In a subset of 100 lactating women, to estimate: 
a. Salt intake of lactating women, their male partners (if applicable), and their 
children 24-59 months (if applicable) using observed weight salt intake 
records 
b. Sodium intake using 24 hr urinary sodium concentrations in lactating women 
5. To test for differences between the 0 mg and 10 mg treatment groups on the following 
outcomes at 24 and 52 weeks postpartum: 
a. Composite and 5 subscales of Mullen 
b. VPC Novelty score and the attention and processing speed subscales 
c. Language Preference Task scores 
d. Oculomotor scores 
6. To determine the effect of inflammation, as measured by C-reactive protein (CRP) 
and α-1-acid glycoprotein (AGP) on the following outcomes: 
a. Maternal ThDP at 2 and 24 weeks postpartum  
b. Infant ThDP at 24 weeks postnatal 
 
3 
STUDY METHODS 
3.1 
Overall Study Design 
Randomised placebo controlled, double-blinded, four-parallel arm, multicentre trial. 
3.2 
Selection of study population 
3.2.1 Inclusion criteria 
Mothers of a newborn who: 
1. are aged 18 – 45 years 
2. had a recent normal pregnancy (i.e. no known chronic conditions, no preeclampsia, 
gestational diabetes etc), and the singleton infant was born without complications (e.g. 
low birth weight (<2.5 kg), tongue tie, cleft palate) 
3. are intending to exclusively breastfeed for six months 
4. reside in Kampong Thom province, and are not planning to move in the next six 
months 

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5. are willing to consume one capsule daily from 2 weeks through to 24 weeks 
postpartum 
6. are willing for her entire household consume only salt provided by the study team 
7. are willing to have study samples collected at 2, 4, 12 and 24 weeks postpartum 
3.2.2 Exclusion criteria 
1. Currently taking or has taken thiamine-containing supplements over the past 4 months 
2. Currently participating in nutrition programs beyond normal care 
3.3 
Treatment groups 
Participants are randomised to receive supplements containing one of the following: 
1. 0 mg thiamine (placebo, negative control group) 
2. 1.2 mg thiamine as thiamine hydrochloride (EAR group) 
3. 2.4 mg thiamine as thiamine hydrochloride (double EAR group) 
4. 10 mg thiamine as thiamine hydrochloride (positive control group) 
The supplements will be packaged in 14-capsule blister packs delivered to participants every 
two weeks. Participants will be asked to consume one capsule per day from 2 weeks to 24 
weeks postpartum. 
3.4 
Method of treatment group assignment and randomisation 
Two product codes for each intervention group (total of 8 product codes), in the form of 3 
letters followed by 8 numbers, were used to assist with blinding. The randomisation schedule 
was produced by the study statistician using ralloc.ado version 3.7.6 in Stata version 15.1. 
Health centre (listed below) was used to define the strata and randomly permuted blocks of 
size 8 were used within strata to assign participants to one of the 8 product codes in the ratio 
1:1:1:1:1:1:1:1. The assignment of product codes to intervention groups was performed by an 
independent individual and kept separate from the randomisation schedule. The 
randomisation was performed using sealed envelopes containing a unique participant ID and 
the assigned product code that were distributed to each health centre. 
Stratification Variable 
Categories 
Health centre 
Tboung Kapoeur Health Centre 
Kampong Svay Health Centre 
Sankor Health Centre 
Chey Health Centre 
Salavisai Health Centre 
Prey Kuy Health Centre 
Prey Pros Health Centre 
Srayov Health Centre 
 

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3.5 
Blinding 
Participants, study staff and study investigators are blinded to treatment group allocation. 
Data analysts will necessarily be unblinded while performing the analysis. 
3.6 
Sample size 
To detect a clinically meaningful difference of 40 μg/L in human milk total thiamine 
concentration between any two treatment groups with 90% power, assuming a SD of 43 µg/L 
(estimated SD of control group in a Cambodian thiamine-fortified fish sauce trial[1]) 48 
women are required per treatment group, or a total of 192 women. This sample size allows 
for 20% attrition and is based on a two-sided alpha of 0.0083 for each of the 6 pairwise 
comparisons between 4 treatment groups to maintain an overall alpha of 0.05. Recruitment of 
320 participants (80 per group) was planned to allow for some uncertainty in the assumed 
values, particularly the SD which may be larger than anticipated. 
Simulations of 500 dose-response curves for human milk total thiamine concentration were 
conducted to estimate the precision that this sample size would provide for addressing the 
primary study objective (i.e. for estimating the dose required to achieve 90% of the average 
maximum human milk total thiamine concentration). Data were simulated based on an 
average minimum and maximum concentration of 136 µg/L[1] and 210 µg/L[2] respectively, 
with a SD of 43 μg/L[1], and reaching 50% and 90% of the maximum average concentration 
at dose 1.2 mg/d (EAR group) and 2.4 mg/d (double EAR group) respectively. Assuming a 
dose-response Emax curve, the precision of the estimated dose is ±1.29 mg/d (i.e. the 95% 
confidence interval for the estimated dose will be within ±1.29 mg/d of the point estimate). 
 
4 
SEQUENCE OF PLANNED ANALYSIS 
4.1 
Interim analysis 
There are no planned interim analyses for this study. 
4.2 
Final analysis and reporting 
Once data collection and cleaning is complete, the database will be locked and unblinded 
treatment codes will be included in the database; a blinded analysis is not possible for 
estimating dose response curves.  
Due to the time-consuming nature of coding video-recorded cognitive assessments, data for 
the following assessments will not be included in the database before it is locked and 
unblinded: primary engagement task, secondary engagement task, Visual Paired Comparison 
Task, and Language Preference Task. Note that the research assistants coding the videos for 
these assessments will remain blinded. 
Analysis of all primary and secondary outcomes will be performed as described in this SAP 
and the results of the statistical analyses will be made available to the Principal Investigator. 
Any post-hoc, exploratory analyses which were not specified in this SAP will be clearly 
identified in the final report.  

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5 
GENERAL ISSUES FOR STATISTICAL ANALYSIS 
5.1 
Analysis software 
All analyses will be performed using R version 3.4.4 or later. 
5.2 
Analysis approach 
The primary analyses will be performed using an intention-to-treat (ITT) approach, where 
analyses will be conducted according to the randomised treatment group regardless of 
participants’ compliance with the protocol. Secondary ‘per-protocol’ analyses will be 
performed including only women who consumed ≥80% capsules over the 22-week 
intervention period. 
5.3 
Methods for withdrawals, missing data and outliers 
All available data collected up until time of withdrawal will be included in the statistical 
analysis.  
Multiple imputation using chained equations will be performed separately by randomised 
treatment group to address missing data under a missing at random assumption. A total of 
100 imputations will be performed to ensure <1% loss of power compared to full information 
maximum likelihood[3]. Covariates pre-specified for adjustment in the analysis will be 
included in the imputation model, along with other auxiliary variables useful for improving 
the prediction of missing values. Conclusions will be based on the results obtained using 
imputed data, with sensitivity analyses performed on unimputed data. 
Outliers will not be excluded from the primary analysis unless a data entry error is confirmed. 
5.4 
Protocol violations and deviations 
No participants will be excluded from the primary intention-to-treat analyses due to protocol 
deviations. 
For the secondary ‘per-protocol’ analyses, only women who consumed ≥80% capsules over 
the 22-week intervention period will be included. 
5.5 
Data transformations 
No data transformations are planned. The statistical analyses detailed in Section 7 are based 
on assumptions about the distribution of the outcomes. If these assumptions turn out to be 
invalid, data transformations may be required. Log transformation is expected to be required 
for analysis of Oculomotor score. 
5.6 
Covariates for adjustment 
Both unadjusted and adjusted analyses will be performed for some outcomes. Conclusions for 
these outcomes will be drawn from the adjusted analyses, with unadjusted analyses 
performed for completeness and to potentially confirm the results of the adjusted analyses. 

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All adjusted analyses will be adjusted for health centre, since it was used as a stratification 
variable in the randomisation process. Adjustment will also be made for additional variables 
collected at 2 weeks postnatal which are potential confounders for some outcomes and these 
have been identified a priori (see Section 7). 
If convergence is an issue, some potential confounders may be excluded from the adjusted 
analysis. Any deviation from the planned adjustment covariates will be documented in the 
final report. 
5.7 
Planned subgroup analyses 
For the secondary outcomes of maternal and infant ThDP and TKac, secondary analyses will 
be performed to test for evidence of effect modification by presence/absence of genetic 
hemoglobin disorder. 
Analysis of secondary outcomes on Mullen, VPC Novelty Scores, Language Preference Task 
scores, and oculomotor scores will be performed in a subset of women comparing only those 
who were assigned to receive 0 mg/d (placebo) to those who were assigned to receive 10 
mg/d (positive control) of thiamine. 
 
5.8 
Multiple comparisons and multiplicity 
Since there is only one primary outcome for the trial and less importance will be placed on 
the results of the secondary outcomes and the secondary analyses testing for interactions, no 
adjustment will be made for multiple outcomes and analyses being performed.  
Since conclusions will be drawn based on the adjusted results (where performed) using the 
imputed data, no adjustment will be made for the fact that both adjusted and unadjusted 
analyses are being performed (where applicable) on both raw and imputed datasets. 
For secondary outcomes where the objective of the analysis is to compare the four treatment 
groups, multiple comparisons will be addressed by first testing whether there is any evidence 
of a treatment group effect. Where this test reaches statistical significance (P<0.05), post-hoc 
tests will be performed to test for differences between each pair of treatment groups and 
Tukey’s honestly significance difference (HSD) will be used to control the type I error rate 
across the 6 pairwise tests.  
 
 
 

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6 
DESCRIPTIVE STATISTICS 
6.1 
Participant flow 
 

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6.2 
Baseline characteristics (2 weeks postnatal) 
A descriptive comparison of the randomised groups will be conducted on all baseline 
characteristics in the 2 weeks postnatal questionnaire. Iron biomarkers ferritin and soluble 
transferrin receptor, vitamin A marker retinol binding protein, and C-reactive protein (CRP) 
and α-1-acid glycoprotein (AGP) at baseline will also be included. Means and standard 
deviations, or medians and interquartile ranges will be reported for continuous variables. 
Frequencies and percentages will be reported for categorical variables. 
 
6.3 
Measurement of treatment compliance 
Treatment compliance will be assessed descriptively by treatment group based on the number 
of unused capsules (Module 1.2 from Fortnightly monitoring data collection sheet) counted 
every fortnight over the 22 week intervention period. 
6.4 
Missing data 
Missing data will be assessed descriptively by treatment group for each outcome variable and 
potential confounder. 
6.5 
Randomisation errors 
Information listed below will be summarised descriptively by treatment group: 
• Number of mothers randomised in the wrong stratum 
• Number of mothers given the wrong treatment 
• Number of mothers discovered to be ineligible after randomisation 
 
7 
STATISTICAL ANALYSES 
In this section, the following details are provided for each outcome variable that will be 
analysed to address the study objectives specified in Section 2: 
• Outcome: A detailed description of the outcome variable, including the type of 
variable and how it will be calculated (if applicable). 
• Analysis: The type of statistical analysis to be performed and parameters to be 
estimated. 
• Adjustment: Where relevant, the covariates at 2 weeks postnatal (stratification 
variables and potential confounders) to adjust for in the adjusted analysis. 
For each outcome variable, parameter estimates will be accompanied by 95% two-sided 
confidence intervals and statistical significance will be assessed at the 0.05 level using a two-
sided test, unless otherwise specified.  
 

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7.1 
Emax curve 
Analysis of the primary and several secondary outcomes will be based on the Emax curve. 
The Emax curve is defined as: 
! = #$ + #&'( )
dose.
#/0$
. + dose.1 
where #$ is the baseline concentration without supplementation, #&'( is the maximum effect 
of supplementation, #/0$ is the dose that produces half of the maximum effect of 
supplementation and corresponds to the inflection point at which the effectiveness of higher 
doses starts to decrease, and ℎ is the slope or Hill factor.  
The nonlinear Least squares model for the primary outcome will be formulated as: 
! = #$ + #&'(
dose.
#/0$
. + dose. + 4 
For secondary outcomes involving testing for effect modification by presence or absence of 
genetic hemoglobin disorder (GD), additional coefficients (5, 6, 7 and 8) will be added to all 
parameters of the Emax curve. The model will be formulated as: 
! = (#$ + #$6GD) + (#&'( + #&'(7GD)
dose(.=.>?@)
(#/0$ + #/0$5GD)(.=.>?@) + dose(.=.>?@) + 4 
Evidence of curve shape differences between presence or absence of genetic hemoglobin 
disorder will be determined by global Likelihood ratio tests. Further testing of the four 
additional coefficients, in the form of A$: coefficient = 0, will be conducted following 
evidence (P<0.05) from the global test.  
The parameter of interest for this study is the dose required to achieve 90% of the maximum 
concentration (i.e. 90% of #$ + #&'() and this will be estimated from the fitted Emax curve 
with variance estimated from non-parametric bootstrapping of 100 resamples. For outcomes 
where there is evidence of curve difference by genetic hemoglobin disorder status (P<0.05 for 
likelihood ratio test), separate estimates for the dose required to achieve 90% of the 
maximum concentration with and without genetic hemoglobin disorder will be reported. 

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7.2 
Primary outcome variable 
7.2.1 Human milk total thiamine concentration (24 weeks) 
Outcome: 
Continuous outcome of total thiamine concentration (µg/L) in maternal 
human milk at 24 weeks postpartum.  
Analysis: 
Fit an Emax curve using nonlinear least squares approach. Estimate of 
the dose that reaches 90% concentration (with 95% CI) will be reported. 
 
7.3 
Secondary outcome variables 
7.3.1 Infant thiamine diphosphate concentration (24 weeks) 
Outcome: 
Continuous outcome of infant thiamine diphosphate concentration 
(ThDP; nmol/L) at 24 weeks postpartum.  
Primary analysis: 
Fit an Emax curve using nonlinear least squares approach. Estimate of 
the dose that reaches 90% concentration (with 95% CI) will be reported. 
Secondary analysis: 
Fit an Emax curve using nonlinear least squares approach allowing 
curve to vary by presence/absence of genetic hemoglobin disorder. 
Estimates of the dose that reaches 90% concentration by genetic 
hemoglobin disorder status will be reported following evidence from 
the global test. Second, test for a linear relationship using linear 
regression with CRP at 24 weeks and treatment group as predictors. 
Estimate of the mean change (with 95% CI) in ThDP for one unit 
increase in CRP will be reported. Third, test for a linear relationship 
using linear regression with AGP at 24 weeks and treatment group as 
predictors. Estimate of the mean change (with 95% CI) in ThDP for one 
unit increase in AGP will be reported. 
Adjustment: 
Health centre (linear regressions only) 
 
7.3.2 Human milk total thiamine concentration (4 weeks and 12 weeks) 
Outcome: 
Continuous outcome of total thiamine concentration (µg/L) in maternal 
human milk at 4 weeks and 12 weeks postpartum.  
Primary analysis: 
Fit a separate Emax curve for 4 and 12 weeks using nonlinear least 
squares model. Estimate of the dose that reaches 90% concentration 
(with 95% CI) at each timepoint will be reported. 
Secondary analysis: 
Fit a linear mixed effects model to outcome at 4, 12, and 24 weeks with 
treatment group, time, and treatment by time interaction as predictors. 
Treatment effects (difference in mean total concentration) will be 
estimated separately for each time point if the interaction p-value is 
<0.05 and for both time points combined otherwise. 
Adjustment: 
Health centre, human milk total thiamine at 2 weeks postnatal (linear 
mixed effects model only) 
 

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7.3.3 Infant transketolase activity coefficient (24 weeks) 
Outcome: 
Continuous outcome of infant transketolase coefficient (TKac) at 24 
weeks postpartum.  
Primary analysis: 
Fit an Emax curve using nonlinear least squares approach. Estimate of 
the dose that reaches 90% concentration (with 95% CI) will be reported. 
Secondary analysis: 
Fit an Emax curve using nonlinear least squares approach allowing 
curve to vary by presence/absence of genetic hemoglobin disorder. 
Estimates of the dose that reaches 90% concentration by genetic 
hemoglobin disorder status will be reported following evidence from 
the global test. 
 
7.3.4 Maternal thiamine diphosphate concentration (24 weeks) 
Outcome: 
Continuous outcome of maternal thiamine diphosphate concentration 
(ThDP; nmol/L) at 24 weeks postpartum.  
Primary analysis: 
Fit an Emax curve using nonlinear least squares approach. Estimate of 
the dose that reaches 90% concentration (with 95% CI) will be reported. 
Secondary analyses: 
First, fit an Emax curve using nonlinear least squares approach allowing 
curve to vary by presence/absence of genetic hemoglobin disorder. 
Estimates of the dose that reaches 90% concentration by genetic 
hemoglobin disorder status will be reported following evidence from 
the global test. Second, test for a treatment effect using linear regression 
and estimate a difference in mean concentration. Third, test for a 
treatment effect using linear regression with treatment group and 
treatment by genetic hemoglobin disorder interaction. Treatment effects 
(difference in mean concentration) will be estimated separately by 
genetic hemoglobin disorder where the relevant interaction term has a 
p-value <0.05. Pairwise comparisons will be reported following 
evidence of a treatment group effect (see Section 5.8). Fourth, test for a 
linear relationship using linear mixed effects model with CRP at 2 and 
24 weeks postnatal, time and treatment group as predictors. Estimate of 
the mean change (with 95% CI) in ThDP for one unit increase in CRP 
will be reported. Fifth, test for a linear relationship using linear mixed 
effects model with AGP at 2 and 24 weeks postpartum, time and 
treatment group as predictors. Estimate of the mean change (with 95% 
CI) in ThDP for one unit increase in AGP will be reported. 
Adjustment: 
Maternal ThDP at 2 weeks postpartum, health centre (linear regressions 
and mixed effects models only) 
 
7.3.5 Maternal transketolase activity coefficient (24 weeks) 
Outcome: 
Continuous outcome of maternal TKac at 24 weeks postpartum.  
Primary analysis: 
Fit an Emax curve using nonlinear least squares approach. Estimate of 
the dose that reaches 90% concentration (with 95% CI) will be reported. 

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Secondary analyses: 
First, fit an Emax curve using nonlinear least squares approach allowing 
curve to vary by presence/absence of genetic hemoglobin disorder. 
Estimates of the dose that reaches 90% concentration by genetic 
hemoglobin disorder status will be reported following evidence from 
the global test. Second, test for a treatment effect using linear regression 
and estimate a difference in mean TKac. Third, test for a treatment 
effect using linear regression with treatment group and treatment by 
genetic hemoglobin disorder interaction. Treatment effects (difference 
in mean TKac) will be estimated separately by genetic hemoglobin 
disorder where the relevant interaction term has a p-value <0.05. 
Adjustment: 
Maternal TKac at 2 weeks postpartum, health centre (linear regressions 
only) 
 
7.3.6 Household salt intake 
Outcome: 
Continuous outcome of total salt consumption (g) in one day.  
Primary analysis: 
Descriptive means and standard deviation (overall and by number of 
individuals in the household) of total salt consumption within a day in a 
subset of 100 households. 
Secondary analysis: 
Descriptive means and standard deviation of the difference in salt 
intake, and intraclass correlation coefficient, between two days in a 
subset of 75 households.  
 
7.3.7 Individual salt intake 
Outcome: 
Continuous outcome of individual table salt intake (g) measured 
throughout one day by field staff.  
Primary analysis: 
Descriptive means and standard deviation of salt intake for a subset of 
100 lactating women, their male partners (if applicable), and their 
children 24-59 months (if applicable). 
 
7.3.8 Sodium intake 
Outcome: 
Continuous outcome of urinary sodium concentration (mmol/24 hours) 
in lactating women.  
Primary analysis: 
Descriptive means and standard deviation of urinary sodium 
concentration in a subset of 100 lactating women. 
 
7.3.9 Mullen (composite and 5 subscales) 
Outcome: 
Continuous outcomes of Composite Mullen and the 5 subscales of the 
Mullen at 24 and 52 weeks postpartum.  

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Primary analysis: 
Test for a treatment effect using a separate linear regression model for 
each outcome and estimate a difference in means (positive control 
minus placebo).  
Adjustment: 
Mullen scales at 2 and 12 weeks postpartum and health centre 
 
7.3.10 VPC novelty score and attention and processing speed subscales 
Outcome: 
Continuous outcomes of VPC novelty score and the attention and 
processing speed subscales at 24 and 52 weeks postpartum.  
Primary analysis: 
Test for a treatment effect using a separate linear regression model for 
each outcome and estimate a difference in means (positive control 
minus placebo). 
Adjustment: 
Hammersmith at 2 weeks postpartum and health centre 
 
7.3.11 Language Preference Task score 
Outcome: 
Continuous outcome of Language Preference Task score at 24 weeks 
postpartum.  
Primary analysis: 
Test for a treatment effect using a linear regression model and estimate 
a difference in means (positive control minus placebo). 
Adjustment: 
Hammersmith at 2 weeks postpartum and health centre 
 
7.3.12 Oculomotor scores 
Outcome: 
Continuous outcome of Oculomotor scores at 24 weeks postpartum.  
This outcome is expected to follow a skewed distribution and hence a 
log or other appropriate transformation will be applied prior to analysis. 
Primary analysis: 
Test for a treatment effect using a linear regression model and estimate 
a difference in means (positive control minus placebo). 
Adjustment: 
Health centre 
 
8 
REFERENCES 
1. 
Whitfield, K.C., et al., Perinatal Consumption of Thiamine-Fortified Fish Sauce in Rural 
Cambodia: A Randomized Clinical Trial. JAMA Pediatr, 2016. 170(10): p. e162065. 
 
2. 
Institute of Medicine, The National Academies Collection: Reports funded by National 
Institutes of Health, in Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, 
Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. 1998, National Academies Press 
(US): Washington (DC). 
 
3. 
Graham, J.W., A.E. Olchowski, and T.D. Gilreath, How many imputations are really needed? 
Some practical clarifications of multiple imputation theory. Prev Sci, 2007. 8(3): p. 206-13.