This study was conducted in six communities in the Lahanam zone, Songkhone District, Savannakheth Province, 600 km south of the capital city, Vientiane, Lao PDR. The Health and Demographic Surveillance System (HDSS) was established in this area in 2004 and all the population is registered.
The Lahanam area has a relatively high production of rice, cotton and watermelon, and most villagers have access to adequate amounts of rice. Other foodstuffs are seasonally available from nearby small patchy forests and from the market town (within 10 km of the Lahanam area). Intake levels of fat, calcium, iron, and retinal in the area were found to be very low compared with the recommended dietary allowance of Thailand and the WHO/FAO .
The first complementary food that is used in the area is principally glutinous rice, which is given pre-chewed by mothers to nearly all infants in the first week of their life. This traditional early use of complementary food may decrease or stop the infant's intake of breast milk .
There is only one health centre in the area providing primary health care. This centre serves a population of 7,000 through six auxiliary nurses and an average of two village health volunteers (VHVs) in each village. A malaria control program was successfully executed in all the villages 10 years ago. Each household was categorized into one of two socioeconomic status groups: high (with electricity, improved water source and latrine) and low (lacking one or all of these).
Study design and randomization
The study was a randomized trial. A statistical power analysis (80% power and significance of p = 0.05) showed that to reduce anaemia by 20% (from 41% to 21%) and to improve haemoglobin concentration by 0.56 g/L, and to compensate for 10% loss to follow-up, a sample size of 110 children would be needed in each group.
In an effort to promote community participation, a series of meetings with local authorities (heads of the villages), parents, VHVs, and health centre staff were held before commencement of the study to explain the study objectives. Individual written informed consent was obtained at the time of enrolment from the mothers or legal guardians of all the children involved in the study. The study was approved by the Nagasaki University Ethical Review Board (Japan) and the National Ethic Committee for Health Research of the Ministry of Health in Lao PDR.
From the HDSS database of the National Institute of Public Health (NIOPH), 367 eligible pre-school age children were identified. Inclusion criteria were: (i) age 6 to 53 months at the time of recruitment; (ii) willingness to participate; (iii) receiving complementary food in addition to breast milk; and (iv) apparently healthy. Exclusion criteria were: (i) having fever or any illnesses on the day of enrolment; (ii) baseline level of haemoglobin less than 70 g/L; and (iii) currently receiving iron supplementation. Of the original 367 children who met the criteria, 17 were absent at the time of enrolment, and 14 were excluded because they had infections with fever on the day of enrolment. Therefore, a total of 336 children were enrolled in the study.
Before recruitment began, we used a simple computer program (a random number generator) for the randomization process, which was done by household. We then enrolled the 336 eligible children and randomly allocated them to three groups: a control group (n = 111), a group given twice weekly multiple micronutrients supplementation (n = 115), and a group given daily multiple micronutrients supplementation (n = 111). Children in families with two or more children who fulfilled the requirements to participate in the study were all included and treated as separate cases; however, all the children in one family were allocated to the same group. There were 215 one-child families, 49 families with two children, and three families with four children in the study.
As part of the routine government health services, all children below the age of five years received a single high dose of vitamin A every 6 months, and those aged 24 months or older received a single dose of mebendazole for deworming in the 2 months prior the study. Children who had not received mebendazole were given it during the baseline survey.
Over 24 weeks, the daily group was given MMP supplementation from Monday to Sunday (7 d/wk), and the twice weekly group was given MMP supplementation on Monday and again on Friday. Because of technical and financial constraints, placebo could not be produced locally, so following the normal standard of care in Lao PDR at the time of the study; the control group received the 6-monthly high-dose vitamin A supplementation instead of a placebo. After completing this study, all subjects in the control group received 60 sachets of MMP.
The MMP supplement used in this study was MixMe™ manufactured by DSM Nutritional Products Europe, Ltd., CH 4002 Basel. The nutrients and amounts used in the multi-micronutrient formula are based on the recommendations by UNICEF/WHO/WFP for one recommended dietary allowance of 15 vitamins and minerals. The nutrient content of 1 g of MMP was vitamin A (RE 400 μg), vitamin D3 (5 μg), vitamin E (TE 5 mg), vitamin B1, B2, B6 each (0.5 mg), folic acid (150 μg), niacin (6 mg), vitamin B12 (0.9 μg), vitamin C (30 mg), iron (10 mg), zinc (4.1 mg), selenium (17 μg), copper (0.56 mg), and iodine (90 μg). The MMP was supplied in a single-dose sachet (1 dose = 1 sachet) and one pack contained 30 × 1 g sachets.
The doses of micronutrients used in this study were calculated based on the WHO recommendation on the dosage schedules for iron supplementation to prevent IDA . Indications for supplementation are when the diet does not include foods fortified with iron or when anaemia prevalence is above 40%. It is recommended that children from 6 to 23 months of age should receive 2 mg of iron per kg body weight per day and children from 24 to 59 months of age should receive the same dose, up to a maximum of 30 mg per day, for 3 months.
The dose of iron in the MMP used in this study was 10 mg, which is within the range of daily recommended dosage of iron for young children according to the American Food and Drug Administration. A total of 168 sachets were provided to the daily supplementation group and 48 sachets were given to the twice weekly supplementation group.
The study commenced in February 2009 with focus group discussions and in-depth interviews with mothers and field promoters (VHVs and members of the Lao Women's Union) to gather information on current child feeding practices and on how to introduce and promote MMP supplementation for children. This phase was completed in March 2009 with a baseline survey in all six communities. Mothers or legal guardians were invited to a central setting such as a health centre or temple in each of the villages, and interviews were conducted by the medical staff from the NIOPH. Information was gathered on; (i) family characteristics including mother's education level, working status (working outside or at home), ownership of latrine, and access to improved water sources; (ii) feeding practices, noting the age of starting complementary foods, the type of complementary foods, and number of meals consumed per day; (iii) knowledge of anaemia; and (iv) medical history (occurrence of diarrhoea or cough in the previous 2 weeks and receipt of routine deworming treatment). After this, the MMP supplementation intervention began, lasted for 24 weeks, and ended in October 2009.
Assessment of haemoglobin concentration
At weeks 0, 12 and 24 of the study, capillary blood samples were obtained from a finger prick using aseptic technique; haemoglobin concentrations were measured immediately with a portable battery-operator Hemocue B-Haemoglobin photometer (Hemocue Inc., Angelholm, Sweden) by trained technicians , who were unaware of the allocation of micronutrient supplement. Anaemia was defined as a haemoglobin concentration below 110 g/L.
The height and weight of the infants and children were measured every 4 weeks during the 24-week intervention. A calibrated SECA scale with intervals of 0.1 kg was used to measure weight. An infant length board with a sliding foot board was used to measure the recumbent length to the nearest 0.1 cm of children less than 24 months, and a wooden scale with a sliding head piece was used to measure the standing height of children 24 months and older. Two field workers, who were unaware of which group a child belonged to, completed the measurements in duplicate using standardized WHO procedures. Height for age, weight for age, and weight for height Z-scores were calculated using the WHO Child Growth Standard (WHO Anthro software, version 3.01) .
Training of village health volunteers
A total of 16 VHVs were recruited and given a 1-day training session before the initiation of the study. The benefits, adverse side effects, usage of the MMP supplement (food demonstration), MMP supplement nutrition education and instructions on follow-up using the monitoring form were all explained. The VHVs delivered MMP supplements to the intervention groups on a weekly basis and instructed the mothers on how to administer the dose in a single meal, twice weekly for one group (the TWS group) and daily for the other (the DS group). To ensure that children consumed the entire dose, mothers were asked to mix the MMP supplement with a small amount of the child's food just before consumption.
Monitoring forms (compliance and acceptability)
The VHVs used monitoring forms to record the number of MMP sachets consumed by the children in the two intervention groups, any side effects, and any illnesses that occurred during the study period. For the TWS group, a VHV visited the house every Monday providing one sachet and every Friday giving the other sachet. For the DS group, VHVs visited the house every Monday and provided all seven sachets. The sachets left unconsumed from the previous week were counted on the next Monday's visit. The three health centre nurses collected the monitoring forms weekly from the 16 VHVs. All monitoring forms were checked by the NIOPH supervisor and reviewed by the main researcher on a monthly basis. The total number of empty sachets was used to measure compliance. For the TWS group, good compliance was defined as when children consumed more than 70% of all the MMP sachets provided, and for the DS group when at least five sachets of the MMP supplements provided per week were consumed during the 24 weeks of the study.
Data were analysed using the PASW statistical package, version 18.0 (SPSS Inc., Chicago, IL, USA). Socio-demographic, health, and nutrition characteristics of the study subjects were summarised as mean and standard deviation (SD) for continuous variables and as frequency for categorical variables. Differences in prevalence were tested with the Pearson chi-squared test. Differences in mean haemoglobin concentration and Z-scores between the groups at the beginning and at the end of the intervention were examined using Repeated Measures Analysis of Variance. The McNemar test was used to assess within group differences at three time points in the study subjects with anaemia and one-way ANOVA was used to assess changes in mean haemoglobin concentration in the treatment groups. All the analyses were carried out for all subjects and separately for the children who were anaemic at baseline. Values of p <0.05 were considered to be significant for all tests.