Micronutrient-Fortified Rice Can Increase Hookworm Infection Risk: A Cluster Randomized Trial

Background Fortification of staple foods is considered an effective and safe strategy to combat micronutrient deficiencies, thereby improving health. While improving micronutrient status might be expected to have positive effects on immunity, some studies have reported increases in infections or inflammation after iron supplementation. Objective To study effects of micronutrient-fortified rice on hookworm infection in Cambodian schoolchildren. Methods A double-blinded, cluster-randomized trial was conducted in 16 Cambodian primary schools partaking in the World Food Program school meal program. Three types of multi-micronutrient fortified rice were tested against placebo rice within the school meal program: UltraRice_original, UltraRice_improved and NutriRice. Four schools were randomly assigned to each study group (placebo n = 492, UltraRice_original n = 479, UltraRice_improved n = 500, NutriRice n = 506). Intestinal parasite infection was measured in fecal samples by Kato-Katz method at baseline and after three and seven months. In a subgroup (N = 330), fecal calprotectin was measured by ELISA as a marker for intestinal inflammation. Results Baseline prevalence of hookworm infection was 18.6%, but differed considerably among schools (range 0%- 48.1%).Micronutrient-fortified rice significantly increased risk of new hookworm infection. This effect was modified by baseline hookworm prevalence at the school; hookworm infection risk was increased by all three types of fortified rice in schools where baseline prevalence was high (>15%), and only by UltraRice_original in schools with low baseline prevalence. Neither hookworm infection nor fortified rice was related to fecal calprotectin. Conclusions Consumption of rice fortified with micronutrients can increase hookworm prevalence, especially in environments with high infection pressure. When considering fortification of staple foods, a careful risk-benefit analysis is warranted, taking into account severity of micronutrient deficiencies and local prevalence of parasitic infections. Trial Registration ClinicalTrials.gov NCT01706419

Rice fortification has been shown to be safe and effective in improving micronutrient status in many studies. However, most studies have been done in Latin America, Africa and India. There are no studies available from Cambodia, except for an acceptability study of fortified rice conducted by WFP and the Institute of Research for Development (IRD) in 2010 in Kampung Speu. This acceptability study showed that fortified rice has an excellent acceptability among school children, parents and teachers. However, there is no data available on whether fortified rice can reduce the prevalence of anemia in school children in Cambodia, nor whether there will be additional benefits, such as better learning capability of school children or fewer days missed due to better health.
To inform WFP, Cambodian policy and the public on the potential benefits of fortified rice on health and development for Cambodian school children, an intervention study will be conducted in Kampung Speu province during the 2012 -2013 school year. The study will be conducted by IRD, PATH and WFP. Sixteen (16) schools will be selected to receive either the normal rice provided by the WFP school meal program, or fortified rice instead of normal rice. Three (3) different types of fortified rice will be tested, to identify the best type of fortified rice. In addition, 4 schools with no school meal program will be selected to serve as control. School attendance and morbidity will be followed in all children in the participating schools over the whole school year. Biochemical indicators of micronutrient status will be determined in a subgroup of children (25% of the children), which requires collection of blood, urine and stool samples.
The study will be submitted to the Ethical Committee of the Ministry of Health for approval, and the Ethical Review Board of PATH, USA. Furthermore, approval will be obtained from the Ministries of Health, Education and Planning prior to the commencement of the study.
Potential benefits of the study includes the immediate treatment of school children found to have severe anemia (hemoglobin < 70 g/L) and improved health and cognitive development for all children receiving fortified rice. Potential disadvantages and risks of the study include side effects of blood taking in the subgroup of children and the disruption of classes for a few days during the data collection.
If the results of the study show improved health and cognition for school children receiving fortified rice, provision of fortified rice to all 500,000 Cambodian school children taking part in the WFP school meal program in Cambodia could become a priority.

FORISCA PROJECT DESCRIPTION
1.1 Introduction More people are affected by anemia and iron deficiency (ID) than by any other micronutrient deficiency, with an estimated 1.6 billion people being anemic and even more people currently having insufficient iron stores 1 . Iron deficiency will eventually lead to irondeficiency anemia (IDA). Indeed, the World Health Organization (WHO) estimates that roughly twice as many people are affected by iron deficiency than by IDA 2 . However, before anemia occurs, iron deficiency is already affecting other functions, such as the immune system and the nervous system, leading to reduced immunocompetence, decreased physical activity and cognitive impairment 3 . On the other hand, many cases of anemia are not due to ID but to other causes such as nutritional deficiencies other than iron (e.g. deficiency of vitamin B12, folic acid or vitamin A), genetic traits (hemoglobinopathies such as sickle-cell or thalassaemia) or chronic inflammation. Besides iron, deficiencies of other micronutrients such as vitamin A, zinc, folic acid and iodine are often highly prevalent in developing countries, including Cambodia 4 . Indeed, micronutrient deficiencies often coexist, and finding only a single micronutrient deficiency in one subject is rather the exception than a rule 5 . Many of these micronutrients function in the same biochemical pathway. Therefore, providing multiple micronutrients instead of only one works synergistically 6 , as for example vitamin A stimulates the production of new red blood cells, improving the utilization of the provided iron 7,8 , and both iron and vitamin A deficiency might aggravate iodine deficiency 9 .
Deficiency of many of these micronutrient deficiencies can also result in delayed physical and cognitive development, resulting in retarded growth and stunting 10 , and in preventing children from reaching their full potential as micronutrients play an important role in children's cognitive and motor development 11 . Indeed, children living in areas affected by severe iodine deficiency disorder (IDD) may have an intelligence quotient (IQ) of more than 12 points below that of non-iodine-deficient areas 12 . Besides iodine, deficiencies of iron and zinc have been associated with impaired psycho-motor development and cognitive function 13,14 . Iron deficiency in school-aged children, even before the onset of anemia, may affect learning abilities 15 and zinc deficiency has been linked with low activity and depressed motor development among the most vulnerable children 16 . As deficiency of several micronutrients has been implicated in impaired cognitive and motor performance and development, correction of a single deficiency may not be enough to substantially improve cognitive performance.
Another factor affecting nutritional status, health and cognitive function is parasite infestation. The exact role of intestinal parasite infection in cognitive development is unclear, as most data is based on cross-sectional surveys which might be biased by factors such as poverty and poor nutritional status 17 . But intestinal parasites reduce the available iron in the gut, and cause local inflammation, thereby causing a loss of nutrients. And frequent absenteeism from school because of illness due to intestinal parasites will affect 1 April 2012 educational achievements for sure 18 . For Cambodia, there is no national data on the prevalence of intestinal parasite infection, but small studies show an infestation rate of >50% 19 . Currently, >75% of the Cambodian school children receive deworming treatment every 6 months, but this might not be often enough to make a lasting impact. Recently, we showed in Vietnam that improving micronutrient status of school children through fortified biscuits markedly reduced the re-infection rate with parasites. Parasite infection rates in children receiving deworming, but no micronutrients were back to baseline prevalence after 6 months, whereas in children receiving both, re-infections rates were lower 20 .

Situation in Cambodia
While Cambodia has seen considerable improvement in access to food and nutritional status since it emerged from civil war in the 1990s, many people continue to suffer from malnutrition and food insecurity and related indicators remain among the lowest in the region. Cambodia ranks 124th of the 169 countries in the 2010 Human Development Index rankings, with 26 percent of its 14.8 million people living on less than $1.25 per day. Over 2.5 million people live in extreme poverty and face serious food deprivation, and the poverty rate for children under five years of age is 38%. The worldwide increase in food prices has also affected Cambodia, accentuating the vulnerability of low-income households. Indeed, child undernutrition in Cambodia is high, with Cambodia being ranked among the 36 countries with the highest burden of child undernutrition 21 .
A recent survey highlighted the need for urgent action, with 11 percent of children under the age of five having severe acute malnutrition, 29 percent being classified as underweight, and 40 percent of the children being stunted. Worryingly, little improvement has been seen in the rates of underweight , stunted, and wasted children since 2005. Therefore, new strategies to combat micronutrient deficiencies in Cambodia are therefore urgently needed. Despite the dearth of data, given the high rates of undernutrition and stunting, micronutrient deficiencies are highly prevalent in Cambodia.

Proportion of undernourished children in Cambodia in the National
The World Food Program (WFP) school feeding program, supported in part through the McGovern Dole program, provides a daily breakfast using a standard nutritionally optimized WFP food ration to almost 500,000 school children in Cambodia. The program is an existing large-scale McGovern-Dole supported intervention with well-established relationships with the government and delivery infrastructure. The program encourages poor and hungry children to come to school and affords them an opportunity to concentrate on their study. In addition, poorer students, particularly girls in the upper primary grades, are provided with a family take home ration.
An assessment of the program found that school feeding was a strong incentive for parents to send their children to school, but only while schools benefited from the program. The evaluation also noted that inclusion of fortified rice could benefit the program if it could reduce the prevalence of iron-deficiency anemia in both sexes. One of the evaluation's final recommendations was that the country office should design and implement modalities for including fortified food in its school feeding program 22 .
Fortification of staple foods is a cost-effective tool to improve micronutrient status of populations at risk for micronutrient deficiencies. Of the tools currently available to alleviate nutritional deficiencies among vulnerable groups, food fortification is the most promising and cheapest 23 . A number of fortified food products are available in Cambodia. However, other than iodized salt, none have reached any significant scale within the country. Rice is the main staple for Cambodians, accounting for 70 percent of daily calorie intake 24 . Per capita rice consumption was 189 kilograms in 2005, which is among the highest intakes of rice in the world. Given this very high consumption rate, the potential impact of fortifying rice in the country could be huge. The current intervention study aims to show the benefits of introducing fortified rice in the regular WFP school meal program on health and development of Cambodian school children.

Objectives and hypotheses Objectives
The main objective of the intervention is to measure the impact of introducing fortified rice in the regular WFP school feeding program on several outcomes, namely: 1. Prevalence of anemia 2. Anthropometry (weight, height, Body Mass Index) 3. Prevalence of micronutrient deficiency (vitamin A, iron, zinc, vitamin B12 and iodine) 4. Cognitive function (as measured with Raven's Colored Matrices and other tests) 5. School attendance (as measured by days of absence due to illness) 6. Parasite infestation and gut flora 7. Immune function (as measured by morbidity recall and ex vivo cytokine responses) Main objectives are objective 1 -5, whereas objective 6 and 7 are secondary objectives, meaning that these last objectives will be included when budget and logistics allow.

Hypothesis
The main hypothesis is that the introduction of fortified rice will decrease: a. the prevalence of anemia (obj. 1) b. the prevalence of micronutrient deficiencies (obj. 3; vitamin A, iron, zinc, vitamin B12 and iodine).
These are direct measurements of micronutrient status, and are sensitive to an intervention. Therefore, a smaller sample size is needed (see below). However, as micronutrient status affects several functional outcomes, the secondary hypothesis is that the improved micronutrient status will also result in better functional outcomes. Specifically, that introduction of fortified rice will result in c. higher anthropometrical indices (obj. 2) d. better cognitive function (obj. 4) e. higher school attendance (obj.5).
These functional outcomes are affected by other factors as well, and therefore less sensitive to an intervention. For these outcomes, a bigger sample size is needed (see below).
In addition, it is hypothezed that consumption of rice fortified with multiple micronutrients will affect immune function, resulting f. increased benefit of half-yearly deworming through lower parasite re-infestation rates (obj. 6) g. Changes in gut flora, harvesting less pathogenic bacteria (obj. 6) h. Increased ex vivo cytokine responses after stimulation (obj. 7) As these outcomes require collection of biochemical data, the sample size for the primary hypothesis will be followed.

Participants, Design and Methods
The study will be a cluster randomized, double blinded, placebo-controlled large scale effectiveness study. Cluster units will be schools in Kampung Speu province, Cambodia, which will be randomized to different treatment (see below). The study sample will consist of school children aged 7-14 years enrolled in participating schools. KampUng Speu province has been selected because it includes schools that both do and do not received support from WFP for the school meals program, giving easy access to a control group. It is also the site of the recently conducted acceptability study which showed excellent acceptability of fortified rice. Moreover, it is not a risk area for flooding, minimizing the risk for logistical problems for the study. During the heavy floods of 2011, school meal programs in some schools in other provinces such as Siem Reap could only be started several months after the school year had started. Finally, it is located within easy access of Phnom Penh which will facilitate monitoring and supervision of intervention activities and transport of biological samples.

Site description.
Kampung Speu is one of the 23 provinces of Cambodia, situated 60 km west of the capital Phnom Penh. Kampung Speu province is subdivided into 8 districts (Baset, Chbar Mon, Kong Pisei, Aural, Udong, Phnom Sruoch Samraong Tong, Thpong), with Kampung Speu town as provincial capital. The majority of the inhabitants are dependent on agriculture for their living, with rice being the main crop. The 2008 population census showed that 716,000 people lived in Kampung Speu, with an annual growth rate of 1.79 and a population density of just over 100 persons/km 2 . Infant mortality in Kampung Speu is still high at 65 / 1000 live births, compared to 13 / 1000 live births in the capital Phnom Penh (2010 CDHS), although primary health care facilities are good, and 89% of children receive full vaccination coverage during their first year of life. Twenty five percent of the population of Kampung Speu is between 5 and 14 years of age, comprising 180,000 children.

Study design
The effectiveness study will be conducted in 20 schools in Kampong Speu province (covering approximately 10,000 children), 16 of which participate in the WFP school meals program, and 4 which are not part of the school meal program. From the 309 schools in Kampung Speu, 90 schools receive school meals, of which 43 receive a school meal in the morning only, that is, there is no shift between morning and afternoon classes. From these 43 schools, 16 will be randomly selected for the study. In addition, from the 34 school not receiving school meals or a take-home ration, 4 schools will be randomly selected. Selection will be done by numbering each school and generation of 2 random lists of 16 schools and 4 schools respectively. The 16 schools receiving school meals will be randomized to receive either the normal school meal food basket (Control group), the original fortified rice (UR_original, the improved fortified rice (UR_improved), or the hot-extruded fortified rice (DSM). The 4 schools not receiving a school meal will serve as double control, to assess the impact of the normal school meal program, and the additional benefits of including fortified rice. The standard WFP school meal consists of rice, canned fish, vitamin-A fortified vegetable oil, yellow split peas, and iodized salt. In the 3 intervention groups with fortified rice, the normal rice shall be replaced with either original Ultra Rice or the improved Ultra Rice, or the fortified rice provided by DSM (NutriRice).
The intervention will run throughout the whole school year, from October 2012 until June 2013. The start of the intervention will coincide with the start of the school year, in October 2012, and run until the end of the school year, 10 months later. The study will involve four groups, each comprising four schools. WFP participating schools will be randomly selected to receive either A control group will be formed from an additional four schools not taking part in the WFP school meal program. During the whole intervention, breakfast will be served to school children each school day (Monday through Saturday).

Composition of the fortified rice
The fortified rice will provide between 30% and 50% of the daily recommended allowance (RDA) of the included vitamins and minerals. In contrast to for example 200,000 IU high dose vitamin A supplements, which are being given to children between 1 and 5 years of age only every 6 months, the aim of fortified rice is to provide a low dose of vitamins and minerals every day. The advantage of this approach is that it is a more physiological approach to improve nutritional status. Moreover, there is no risk of toxicity by providing too much of a vitamin or mineral.
There are several methods for producing fortified rice. Two widely used methods are extrusion and waxing.
With the extrusion method, artificial rice kernels are made from a mixture of rice flour, a premix of vitamins and minerals and a binding agent. Extrusion can be cold (temperatures <70C) or hot (temperatures between 70C and 110C). The artificial rice kernels are mixed in a ratio of 1:100 with normal rice, making fortified rice suitable for consumption. Waxing uses a coat with a premix of vitamins and minerals which are spread over the rice kernels. As washing of the rice before cooking might remove the wax coat, and with this, the vitamins and minerals, waxing is considered not suitable for SE Asia. 1 April 2012 The FORISCA study will compare 3 different types of fortified rice, made with the extrusion method. The first type of rice is the original fortified rice from PATH. This type of rice does not contain vitamin A, and the fortified rice kernels are produced with cold extrusion. The second type of rice is the improved fortified rice kernel from PATH, which does contain vitamin A. As vitamin A and iron can react together, the vitamin A and iron have been coated before mixing with the rice flour. The temperature of extrusion is just above 70C, so the method of extrusion is somewhere between cold and hot. The final type of fortified rice kernel will be from DSM (NutriRice), which is a hot-extruded fortified rice kernel containing vitamin A and iron. For complete composition of the rice kernels, please see below.
The table below summarizes the aimed fortification levels for the different vitamins and minerals of the 3 types of fortified rice used in the FORISCA project, as well as the toxicity levels. Recruitment of participants for the in-depth study.

Vitamin / Mineral
As described above, schools will be randomly selected from a list of all eligible schools in Kampung Speu province. Once schools have been selected, schools will be informed of the study, and a complete list of names of the children attending the school will be requested, as well as their class and gender. From this list, 150 children per school will be randomly selected, stratified for gender and class. As we expect that ~ 10% of the children will not be available (e.g. moved out of the area over the summer holidays), for each school we will generate a list of 180 children (20% excess). The school monitor will visit all homes of the children in the month before the school starts to obtain written informed consent of the parent for the in-depth study, and the first 150 children who will be present after the school holidays and whose parents agree to the study will be selected from each school.

Ethical consent of all participants.
During the first school week, information meetings will be organized for parents of all children, in which parents will be informed of the study, and that data on absenteeism and morbidity of their children will be recorded during the whole year. All parents will be asked for a signed informed consent form. Data will not be collected from children whose parents do not want to participate in the study.
(please see attached informed consent forms: 1x in-depth and 1x general)

Data collection and usage
Data on school attendance and reason for absence and morbidity will be collected from all children in the participating schools on a daily basis. This data will be used to assess the impact of the school meal program and the introduction of fortified rice on days absent and general health. Anthropometrical and cognitive data and biological samples will be collected in the sub-group of children participating in the in-depth study (25% of all children) at baseline (T0), after 5 months (T5, mid-way) and at the end of the school year after 10 months (T10), with the exception of stool samples, as these will be collected more frequently to assess the intestinal parasite re-infection rate. Children for the in-depth study will be selected at random from each school, using a computer list with all children attending the school, and specific informed consent will be obtained from the parents. For the in-depth study, age range will be limited to 8 -13 years, due to the difficulties of obtaining reliable cognitive function tests in younger children, and the limited number of children with an age>13 still in primary school. Anthropometrical data will be used to assess the impact of the school meal program (control) and the introduction of fortified rice (2 intervention groups) on the weight and height gain, and the acquisition of lean body mass (muscle mass). The cognitive data will be used to measure cognitive performance in the children, and to test whether improvements in micronutrient status are accompanied by increases in the ability to develop new insights and information from what is already perceived or known. The biochemical samples will be used to determine micronutrient and immune status (blood and urine samples) and parasite infestation and gut flora (stool sample).
For the implementation of the effectiveness study, monitors will be hired for the whole school year. For each school, 1 monitor will be hired (hence 20 in total). Before the start of the effectiveness study, school monitors will trained in a) providing information of the study and answering questions related to the study, to vitamins and minerals in general and fortified rice; b) monitoring daily school attendance (using a prepared form with all children per class); c) monitoring reason of absence of children by home visit (scheduled for each afternoon after school finishes at noon). In addition to this, there will be 1 overall field coordinator, based in Kampung Speu province, to deal with day-to-day problems and questions.
For the collection of data (anthropometry, cognition) and samples (blood, urine, stool) teams will be formed for anthropometrical assessment, for cognitive function assessment and for biological sample collection. For anthropometry, it is estimated that 1 team of 4 persons can measure 50 school children in 1 morning. Therefore, 6 teams will be formed, to be able to measure 2500 children within 15 days For cognitive function, it is estimated that 1 person will need 30 minutes to test 1 child. Therefore, to make sure that the cognitive team can also assess 50 children / school / morning, 1 team will consist of 8 persons will perform the cognitive assessment, with 6 teams for cognitive assessment in total. For the collection of biological samples, it is estimated that 1 team can obtain samples from 40 school children / day. Hence, 6 teams (of 3 persons) will be formed, to be able to measure 2500 children within 15 days.

Gaining consent
Written informed consent will be obtained from the parents or caretaker of each child participating in the study, as the study will be collecting data on morbidity and absence. In addition, a more elaborate informed consent will be obtained from the parents of caretakers of the children participating in the in-depth study. (See appendix)

Sample size considerations.
Reasons for collection of in-depth data from only a sub-group of children are that biochemical analysis of blood samples is expensive, and the cognitive tests are time consuming. However, as the biochemical analyses are sensitive to changes, a sample size of 500 children per group, is enough to show a significant effect of the fortified rice on hemoglobin concentration if the increase in hemoglobin concentrations is >4 g/L (average hemoglobin concentration 110 g/L). Moreover, the study can show a decrease in the prevalence of anemia of 9% or more (expected prevalence of anemia = 50%). For the other main biochemical indicators, a sample size of 500 children / group is sufficient to show biological significant impact. For objectives 5 (school attendance) and 7 (morbidity), a larger sample size is required to allow meaningful conclusions. Therefore, these outcomes will be followed in all 10000 children.

Anthropometry
Prior to the study, all staff measuring anthropometry will be trained in the correct procedures for obtaining weight, height, arm circumference and skinfolds measurements. Staff will be re-trained before the mid-term and end-point data collection weeks. Weight will be measured on each child wearing light clothing using the Body Composition Monitor Scale from Tanita BC-543, Japan to the nearest 0.1 kg. The accuracy of the scales will be checked every day using a set of calibration weights. Height of the children will be measured by a portable measuring tape (USA) to the nearest 0.1 cm. Height will be measured twice and the average value will be taken. If the two measures vary by more than 3 mm, a new set of two measures is taken until difference between the two is less than 3 mm. Arm circumference measurements (MUAC) will be made using a flexible, non-stretch tape. The measurement is taken at the midpoint of the upper left arm, between the acromion process and the tip of the olecranon. Triceps skinfold will be measured at the midpoint of the back of the upper left arm. All anthropometrical data will be recorded on a specific form which will be checked by the team supervisor daily.

Blood sampling
Blood sampling will be done at the village health centre. From a selected school, children (10/hr) will be brought by car to the village health centre, where a blood, urine and stool sample will be collected by the village health centre staff. Five (5) mL of venous blood will be drawn by venipuncture from the antecubital vein in a trace-element free vacutainer with anticoagulant (Vacuette, Greiner Bio One) by an experienced nurse from the local Health Centre in each village, using standard, sterile procedures. Immediately, 100 µl of whole blood will be used to fill a microcuvette for the determination of hemoglobin concentrations using the HemoCue system (HemoCue Angholm, Sweden). Certified control material will be measured daily prior to starting the measurements. All values of controls and duplicates will be noted and reported. Children with severe anemia (hemoglobin concentration < 70 g/L) will be send to the Provincial Health Centre for further diagnosis and treatment. The costs for transport, diagnosis and treatment will be for the project. The HemoCue Hemoglobin system consists of disposable microcuvettes with reagents in dry form and a single purpose designed photometer. No dilution is required. The photometer is calibrated at the factory against the cyanmethemoglobin method, which is the international reference method for the determination of the total hemoglobin concentration in blood. The remaining blood sample will be stored immediately in the dark in a cool box (4 C), and transported to the laboratory of Provincial Health Center (PHC) within 4h of blood collection. At the Provincial Health Center, whole blood will be centrifuged at 3000g for 10 min at room temperature and the plasma will be divided into five aliquots of 500 µl. Four aliquots of 500 µL will be put into pre-labeled eppendorf tubes and be frozen immediately at -20 C until transported to the laboratory of the National Institute of Public Health (NIPH) in Phnom Penh, where the samples will be frozen at -70°C until analysis. The last aliquot of 500 µl will be put into a 2.5 ml tube containing buffer (RPMI) and stimulant (LPS+PHA), and be incubated for 24 h at 37°C. Thereafter, the tube will be centrifuged and the supernatant collected and stored for -20C until transported to NIPH where after it will be stored at -70 C until analysis of cytokines produced.

Biochemical indicators of micronutrient and immune status Iron status.
Iron status will be determined using plasma concentrations of 2 proteins: ferritin and soluble transferrin receptor (sTfR). Ferritin concentrations are a measure for the amount of iron stored in the body. Concentrations <15 µg/L indicate depleted iron stores. sTfR concentrations are a measure for the demand of iron by the tissues. If iron becomes depleted in the tissue, sTfR concentrations will increase. Using a algorithm total iron body stores can be calculated. Both ferritin and sTfR plasma concentrations will be determined with commercially available ELISA kits (Ramco Inc). Plasma ferritin and transferrin receptor (aliquot 1) will be measured by ELISA using commercial kits (RAMCO) that include reference material, and will be performed at the Laboratory of NIPH. A 20 µL aliquot plasma will be used for the both the ferritin and TfR assay using the ELISA procedure (RAMCO Laboratories, Inc, Houston, Texas). The accuracy will be checked using WHO international standards (Ramco Laboratories).

Vitamin A status.
Vitamin A status is determined by measuring plasma retinol concentrations using a High Performance Liquid Chromatography with UV detection at 325 nm. Plasma retinol concentrations (aliquot 2) will be determined by HPLC (LC 10ADvp, Shimadzu, Japan,) according to the method of the International Vitamin A Consultative Group at the laboratory of the National Institute of Nutrition (NIN), Ministry of Health, Hanoi, Vietnam, using 400 µl of plasma and retinyl acetate as internal standard.

Zinc status.
Although plasma zinc concentrations are not strongly related to individual zinc status, due to the fact that many factors can affect zinc concentrations, plasma zinc concentrations are the best indicator for zinc status on a population level. Plasma zinc concentrations (aliquot 3) will be determined by flame atomic absorption spectrophotometry (spectrophotometer GBC, Avanta) using trace element-free procedures at NIN, Hanoi, Vietnam (2x 200 µl of plasma). Results will be verified against reference materials for zinc. To control for any potential contamination of material used for blood sampling and processing by external zinc, analysis will be carried out of the zinc content of at least 20 sets of material (needle, syringe, vacutainers and eppendorf tubes) when blood will be replaced by twice-distilled, deionized and demineralized water.

Inflammatory and immune status.
C-reactive protein (CRP) and α-1 acid-glycoprotein (AGP) are 2 acute phase proteins which rise early (CRP) and late (AGP) during an infection, under the influence of cytokines such as interleukin-6. CRP and AGP (aliquot 4), are measured to quantify the acute phase response to be able to correct for the effect of inflammation on the indicators described above. CRP, AGP and cytokine concentrations will be determined using commercial avaible ELISA kits (ICL Laboratories) which includes reference samples. 100 µL aliquots of plasma will be used for CRP and AGP assay, with 10% of the samples being done in duplicate. Assays will be done at the Laboratory of NIPH, Phnom Penh, Cambodia. Cytokines (IFN-y, IL-2, IL-6, IL-10, aliquot 5) will be measured as indicators of immune activation using commercially available ELISA kits (ICL Laboratories) at the laboratory of NIPH. Cut-offs for acute and chronic inflammation are a CRP>mg/L and a AGP>1.0 g/L respectively.

Iodine status.
Urinary iodine concentration is a reliable measure for iodine intake of an individual over the last few days. The method used includes digestion of the urine sample with ammonium persulfate, a Sandell-Kolthoff reaction and reading at 340 nm. Urinary iodine concentrations will be measured at the National Institute of Nutrition in Hanoi, Vietnam.

Parasite infestation.
Parasites eggs (ascaris, trichuris, hook worm) will be counted in stool samples. Stool sample will stained and counted within one hour after staining by Kato-Katz method (WHO, 1994). The egg output will be expressed as mean eggs per gram faeces (epg).

Gut flora
Gut flora will be determined using bacterial DNA obtained from the stool samples with a commercial available kit for DNA extraction, using PCR techniques. Gut flora will be determined at the Free University Hospital, Amsterdam, The Netherlands.

Cognitive Function
Raven's Colored Progressive Matrices test will be used to measure the cognitive performance in the children. The test measures the ability to develop new insights and information from what is already perceived or known. The test uses 36 pictures with a pattern, in which children need to identify the missing piece out of a choice of 6 different options. The patterns increase in difficulty. The test is almost free of cultural bias as the test minimizes the impact of language skills and cultural bias and is appropriate for both children and adults. Raven's Matrices measure two complementary components of general intelligence: the capacity to think clearly and make sense of complex data (educative ability) and the capacity to store and reproduce information (reproductive ability). The Colored Matrices are especially suitable for children aged 5 to 11 years. Children will be tested individually by 1 team member of the cognitive team, while seated on a desk. In a separate room, 8 desks and chairs will be positioned with adequate space between them as not to have interference between the children while doing the testing. In addition, a series of cognitive tests will be selected from Wechsler's Intelligence Scale for Children III (WISC III), namely Digit span backward and forward, coding, and block design. The Digit span backward test assesses working memory for auditory information whereas the Digit span forward assesses children's auditory attention span and the ability to focus on auditory information. Coding requires children to quickly pair either shapes or numbers with a symbol and measures the speed of information processing. Block design is a measure of problem solving to assess executive function short-term memory and attention span. These tests were selected based on their wide usage, free of cultural bias and validity. All of these tests have been used successfully in earlier studies in Vietnamese schoolchildren.
The cognitive tests will be conducted by teams of university students who have been trained on the test methods and use of published materials translated into Khmer from the original test. The children will be assigned randomly to the technician. As for the other members of the research team, nobody in the team will be aware of the allocation of the school to control or fortified rice. However, we cannot guarantee that information concerning whether a school meal is served in the school or not (double-control) will be known to the field team. The duration of the whole range of tests is estimated to about 20-30 minutes. Each child will conduct the Raven's Colored Progressive Matrices test first, followed by the other sub-tests of WISC III. Similar procedures will be conducted at baseline and endpoint survey. Because the test has not been standardized locally, interpretations of the scores of cognitive test will be used as raw scores.

Other measurements
In addition to the data collected from the school children, samples from the rice served in each school will be taken at baseline (after the start of the intervention), and every 2 months thereafter. Samples will therefore be taken at t=0, t=2, t=4, t=6, t=8 and t=10. In each school (n=16), 5 samples will be taken from the rice served in the classroom (from 5 different classes). Hence for each time point, 80 samples will be available, and for the whole duration of the intervention 480 samples will be available. These samples will be analyzed for micronutrient content (vitamin A, iron, zinc) at the Institute of Nutrition, Mahidol University, Bangkok, Thailand.

Data management and statistical analyses
A database will be constructed, using double entry of data to improve quality. All original data forms will contain the name of the participant to be able to check for the correct identity during data collection. The database however will be anonymized, with all participants being allocated a number which cannot be traced back to the participant without having the original data form. Original data forms will be stored at the Department of Fishery for up to 10 years after completion of the FORISCA project, and will then be destroyed.

Ethical considerations
Subjects of this intervention study will be schoolchildren from Grade 1 to Grade 6 (between 6 and 13 yrs of age). For the overall study, parents will be informed that their children might or might not receive rice during the whole school year which added vitamins and minerals. The amount of vitamins and minerals added will not exceed 50% of the recommended daily allowance. In addition, data on school attendance and morbidity will be kept. Parents can refuse either to data collection or to participate in the whole study. If parents refuse to have data collected on the attendance and morbidity of their child, the name of the child shall be omitted from the class list, and no information will be recorded. If parents refuse to participate in the study, a separate breakfast meal will be provided for the child. If many parents in a school refuse to participate (>5% of the parents), another school will be selected.
For the in-depth study, specific and detailed informed consent will be obtained. After selection, participants (parents of children and children) will be informed about the purposes and procedures of the study. At least one parent will be asked to sign the consent form presented in the annex. All refusals will be accepted without asking for reason of refusal. All respondents will be free to leave the survey at any time during the study, and this will be explained to the parents also. Before the implementation of the study, extensive consultation will be undertaken with the villages, the districts, and provincial authorities as well with school staff and parents of children. The protocol of the study will be submitted to the Ethical Committee of the Ministry of Health, Kingdom of Cambodia, for approval before implementing the study. Children with severe anemia (Hb < 70 g/L) will be referred to the Provincial Health centre for further diagnosis and treatment. All costs (diagnosis, transport and treatment) will be for the project. Children with mild anemia will not be treated, as the consequences of mild anemia on health are currently unclear.
Possible adverse effects.
The study will collect blood samples using vena-puncture. Besides being painful, possible adverse effects include collapse of the child and phlebitis or other infection at the site of blood drawing. Concerning collapse of the child, this is caused by a strong vaso-vagal nervous reaction, and although harmless in itself, can cause harm if the child is standing. Therefore, children will be seated in a comfortable chair during blood taking. The risk for phlebitis or other infections will be minimized by using sterile techniques and cleaning the area of the blood drawing with 70% alcohol before the actual blood draw. No other possible adverse effects are foreseen.

Data monitoring board.
A data monitoring board will be formed who will review the data after the mid-term (T5) evaluation. Although data on morbidity might not be completely available at that time, data on hemoglobin and anemia prevalence will be available. Given the low incidence of mortality in school-aged children, we expect that there will be no data analysis on mortality.
Mortality data analysis will be done if there are more than 5 cases. School children are vulnerable for a lack of good nutrition (undernutrition) which can lead to a lack of vitamins and minerals (micronutrient deficiencies). Children need vitamins and minerals to stay healthy and to be able to do well at school. We are doing a study, in which we will compare three types of rice that have added vitamins and minerals. Most children in the study (in Kampung Speu) will get rice through the normal school meal program from the World Food Program school meal project.
-If the school of your child is in the program from WFP, this program will continue as normal. However, in some schools, we will replace the normal rice with the fortified rice, that is, the rice with the extra vitamins and minerals.
-If the school of your child is not participating in the program of WFP, also nothing will change. For the coming year, the school will not be included, and there will be no school meal served at school.

Questionnaires
We want to know the benefits of the fortified rice on the health and learning capabilities of the school children. Therefore, we would like to follow the children in the schools for 1 whole school year (from October 2012 until July 2013). We will record the names of the children who are absent each day, and we will come to your house to ask why the child was not at school. Maybe because the child was ill, or maybe for some other reason. We will not give this information to school, but record it only for the study, and it has no consequences for the school meal your child is receiving nor for anything else. If the child was ill, we would like to know what kind of symptoms the child had.

In-depth study
In addition to the questions asked above, we would like to have more detailed information on the nutritional status and learning capacities of the children. For this, we will need to collect blood, stool and urine samples. However, we will not collect these data in all children, only in ¼ of the children in the school. Your child has been selected to be one of those to give blood, urine and stool samples. Blood and urine samples will be collected 3 times over the year (as baseline, after 5 months and at the end of the school year). Stool samples will be collected every 2 months.
The amount of blood taken will be 5 ml, or about one teaspoon of blood from your child's inner elbow. We will test your child's blood for anemia, that is, whether s/he has enough blood cells, and for vitamin and minerals in the blood (vitamin A, iron and zinc, B-vitamins, vitamin D), general infection and inflammation. The blood will not be used for anything else. If we find your child has severe anemia, we will refer him/her to the Health Centre for treatment. All costs for this will be carried by the project, and will not be yours. The stool and urine sample will be used to look for iodine deficiency and for parasites. Your participation is entirely your choice. Whether you choose to participate or not, will not affect the school meal your child might be receiving, nor anything else. Although we hope you will continue with the study for the full 10 month period (October 2012 -July 2013), you can stop participating with the study any time during the study, and only have to tell you that you don't want your child to participate anymore in the study. If at any time during the study you have any questions, you can call this number -(INCLUDE PHONE NUMBER OF STAFF RESPONSIBLE) We would like to ask for your participation in the study now. If you agree to participate with your child in the study, please sign or mark your mark in the box below.
Signature or thumbprint of the caregiver: For study fieldworker I have read the consent form in its entirety to the caregiver of the child.
Signature of study fieldworker: _____________________________________________________ Date (day/month/year): _______/_______/________ Leave a copy of the consent form with the caregiver. Circle the telephone number on the page to ensure that they understand they can call for more information.

Ethical considerations
Subjects of this intervention study will be schoolchildren from Grade 1 to Grade 6 (between 6 and 13 yrs of age). For the overall study, parents will be informed that their children might or might not receive rice during the whole school year which added vitamins and minerals. The amount of vitamins and minerals added will not exceed 50% of the recommended daily allowance. In addition, data on school attendance and morbidity will be kept. Parents can refuse either to data collection or to participate in the whole study. If parents refuse to have data collected on the attendance and morbidity of their child, the name of the child shall be omitted from the class list, and no information will be recorded. If parents refuse to participate in the study, a separate breakfast meal will be provided for the child. If many parents in a school refuse to participate (>5% of the parents), another school will be selected.
For the in-depth study, specific and detailed informed consent will be obtained. After selection, participants (parents of children and children) will be informed about the purposes and procedures of the study. At least one parent will be asked to sign the consent form presented in the annex. All refusals will be accepted without asking for reason of refusal. All respondents will be free to leave the survey at any time during the study, and this will be explained to the parents also. Before the implementation of the study, extensive consultation will be undertaken with the villages, the districts, and provincial authorities as well with school staff and parents of children. The protocol of the study will be submitted to the Ethical Committee of the Ministry of Health, Kingdom of Cambodia, for approval before implementing the study. Children with severe anemia (Hb < 70 g/L) will be referred to the Provincial Health centre for further diagnosis and treatment. All costs (diagnosis, transport and treatment) will be for the project. Children with mild anemia will not be treated, as the consequences of mild anemia on health are currently unclear.
Possible adverse effects. The study will collect blood samples using vena-puncture. Besides being painful, possible adverse effects include collapse of the child and phlebitis or other infection at the site of blood drawing. Concerning collapse of the child, this is caused by a strong vaso-vagal nervous reaction, and although harmless in itself, can cause harm if the child is standing. Therefore, children will be seated in a comfortable chair during blood taking. The risk for phlebitis or other infections will be minimized by using sterile techniques and cleaning the area of the blood drawing with 70% alcohol before the actual blood draw. No other possible adverse effects are foreseen. The collection of urine and stool samples does not pose any health risk, although it might give an uncomfortable feeling for some children and/or their parents.

Annex 7 -COMPENSATION MECHANISMS
There will be no financial compensation for any of the children and/or their parents just for participating in the study. All children will receive a school meal, 6 days/week as intended by the WFP's school meal program, or no school meal, when the school they are visiting is not participating in the WFP school meal program. However, medical costs and transport costs will be paid by the project for any child being diagnosed with a hemoglobin concentration of less than 70 g/L (severe anemia). It is expected that ~5% of the children will have severe anemia, for which appropriate medical treatment will be given.