Adherence and acceptability of multiple micronutrient supplementation during pregnancy: Study protocol for a cluster-randomized non-inferiority trial in Cambodia.

BACKGROUND: Iron and folic acid (IFA) supplements are currently provided to Cambodian women during pregnancy. However, recent research has found benefits of a multiple micronutrient supplement (MMS) over just IFA alone on several outcomes of perinatal and infant health. The Ministry of Health in Cambodia has proposed a transition from IFA to MMS but to effectively guide this transition requires implementation research on the acceptability and adherence to MMS (over IFA). METHODS: This non-inferiority trial aims to assess the adherence and acceptability of IFA (60 mg elemental iron and 400 µg folic acid) compared to MMS (standard UNIMMAP formulation including 15 micronutrients) during antenatal care in Cambodia. A prospective cohort of 1545 pregnant women will be assigned to one of three trial arms: (1) IFA for 90 days [IFA-90]; (2) MMS for 180 days with two distributions of 90-count tablet bottles [MMS-90]; or (3) MMS for 180 days with one 180-count tablet bottle [MMS-180]. Each arm will enroll 515 women across 48 health centers (clusters) in Kampong Thom Province in Cambodia. The primary outcome is the non-inferiority of adherence rates of MMS-180 compared to IFA-90, as assessed by tablet counts. Mixed-effects logistic and linear regression models will be used to estimate the difference in the adherence rate between the two groups, with an 'a priori' determined non-inferiority margin of 15%. Acceptability of MMS and IFA will be measured using a quantitative survey conducted with enrolled pregnant women at 30-day, 90-day, and 180-day time-points. DISCUSSION: Findings from this study will guide an effective and feasible MMS scale-up strategy for Cambodia. Additionally, the findings will be shared globally with other stakeholders planning to scale up MMS in other countries. TRIAL REGISTRATION: NCT05867836 ( ClinicalTrials.gov , registered May 18, 2023).

Reticulocyte haemoglobin equivalent (RET-He) as an early marker of responsiveness to oral iron supplementation.

AIM: We investigated the potential of reticulocyte haemoglobin equivalent (RET-He) as an early marker of responsiveness to iron supplementation. METHODS: Data were obtained from a randomised controlled trial of daily iron supplementation in 356 Cambodian women (18-45 y) who received 60 mg elemental iron for 12 weeks. A fasted venous blood specimen was collected at baseline, 1-week and 12-week timepoints. Whole blood haemoglobin (g/L) and RET-He (pg) were measured using a Sysmex haematology analyser. RET-He measures were evaluated for their predictive ability on haemoglobin response to iron supplementation (defined as ≥10 g/L at 12 weeks). Receiver operating characteristic (ROC) curves were used to assess discrimination performance, and the area under the ROC curve (AUCROC) served as a measure of the ability of each predictor to discriminate between women likely or unlikely to elicit a haemoglobin response. RESULTS: Predictive ability (AUCROC (95% CI)) of baseline, 1-week, and change from baseline to 1-week RET-He on haemoglobin response was 0.70 (0.63 to 0.76), 0.48 (0.41 to 0.56) and 0.81 (0.75 to 0.87), respectively. Based on the Youden index, an absolute increase in RET-He of ~1.1 pg or a percentage increase of ~4.4% over 1 week were optimal thresholds to predict responsiveness to iron supplementation. CONCLUSION: Single timepoint RET-He measures have poor predictive ability; however, change in RET-He after 1 week was a strong predictor of haemoglobin response among Cambodian women receiving 60 mg elemental iron and can be measured easily and quickly after only 1 week of iron therapy.

Restricting diet for perceived health benefit: A mixed-methods exploration of peripartum food taboos in rural Cambodia.

Food taboos encompass food restrictions practiced by a group that go beyond individual preferences. During pregnancy and lactation, food taboos may contribute to inadequate nutrition and poor maternal and infant health. Restriction of specific fish, meat, fruits and vegetables is common among peripartum women in many Southeast Asian countries, but data from Cambodia are lacking. In this mixed-methods study, 335 Cambodian mothers were asked open-ended questions regarding dietary behaviours during pregnancy and up to 24 weeks postpartum. Descriptive statistics and content analysis were used to characterize food taboos and multiple logistic regression analyses were conducted to identify predictors of this practice. Participants were 18-44 years of age, all of Khmer ethnicity and 31% were primiparous. Sixty-six per cent of women followed food taboos during the first 2 weeks postpartum, whereas ~20% of women restricted foods during other peripartum periods. Pregnancy taboos were often beneficial, including avoidance of sugar-sweetened beverages, coffee and alcohol. Conversely, postpartum avoidances typically included nutrient-dense foods such as fish, raw vegetables and chicken. Food taboos were generally followed to support maternal and child health. No significant predictors of food taboos during pregnancy were identified. Postpartum, each additional live birth a woman had reduced her odds of following food taboos by 24% (odds ratio [95% confidence interval]: 0.76 [0.61-0.95]). Specific food taboo practices and rationales varied greatly between women, suggesting that food taboos are shaped less by a strict belief system within the Khmer culture and more by individual or household understandings of food and health during pregnancy and postpartum.

High consumption of unhealthy commercial foods and beverages tracks across the complementary feeding period in rural/peri-urban Cambodia.

Consumption of unhealthy commercial foods and beverages (UCFB) is common among infants and young children living in low- and middle-income countries. Such foods can displace other nutritious foods, however, there is limited evidence on how this consumption tracks across time. This study assessed and tracked UCFB consumption of children living in rural/peri-urban Cambodia during the complementary feeding period, identified UCFB consumption patterns of these children, and explored the association between UCFB consumption and growth. A 6-month longitudinal cohort study was implemented among 567 caregivers of children aged 10-14 months at recruitment. UCFB consumption was estimated each month via a telephone-administered 7-day food frequency questionnaire, and UCFB consumption patterns were identified based on changes in this frequency of consumption over time. The majority of children either maintained (45.7%, n = 246) or developed (43.5%, n = 234) an unhealthy consumption pattern and only 10.8% (n = 58) of children maintained/transitioned into a healthy consumption pattern. High consumers of UCFB at 10-14 months had a 4.7 (CI: 4.7 [3.1-7.2]) times odds of being high consumers of UCFB at 15-19 months (p < 0.001). There was a trend of lower length-for-age z-scores (LAZ) among children maintaining or developing an unhealthy consumption pattern (~-0. SD LAZ) compared to children maintaining/transitioning into a healthy consumption pattern, however, this association was not statistically significant. Findings indicate that high UCFB consumption begins during infancy and tracks into early childhood. National policies and programmes centred on early interventions addressing the use of UCFB for infant and young child feeding are needed.

Daily Oral Supplementation with 60 mg of Elemental Iron for 12 Weeks Alters Blood Mitochondrial DNA Content, but Not Leukocyte Telomere Length in Cambodian Women.

There is limited evidence regarding the potential risk of untargeted iron supplementation, especially among individuals who are iron-replete or have genetic hemoglobinopathies. Excess iron exposure can increase the production of reactive oxygen species, which can lead to cellular damage. We evaluated the effect of daily oral supplementation on relative leukocyte telomere length (rLTL) and blood mitochondrial DNA (mtDNA) content in non-pregnant Cambodian women (18-45 years) who received 60 mg of elemental iron as ferrous sulfate (n = 190) or a placebo (n = 186) for 12 weeks. Buffy coat rLTL and mtDNA content were quantified by monochrome multiplex quantitative polymerase chain reaction. Generalized linear mixed-effects models were used to predict the absolute and percent change in rLTL and mtDNA content after 12 weeks. Iron supplementation was not associated with an absolute or percent change in rLTL after 12 weeks compared with placebo (ß-coefficient: -0.04 [95% CI: -0.16, 0.08]; p = 0.50 and ß-coefficient: -0.96 [95% CI: -2.69, 0.77]; p = 0.28, respectively). However, iron supplementation was associated with a smaller absolute and percent increase in mtDNA content after 12 weeks compared with placebo (ß-coefficient: -11 [95% CI: -20, -2]; p = 0.02 and ß-coefficient: -11 [95% CI: -20, -1]; p= 0.02, respectively). Thus, daily oral iron supplementation for 12 weeks was associated with altered mitochondrial homeostasis in our study sample. More research is needed to understand the risk of iron exposure and the biological consequences of altered mitochondrial homeostasis in order to inform the safety of the current global supplementation policy.

Thiamine supplementation holds neurocognitive benefits for breastfed infants during the first year of life.

Women reliant on mostly rice-based diets can have inadequate thiamine intake, placing breastfed infants at risk of thiamine deficiency and, in turn, physical and cognitive impairments. We investigated the impact of maternal thiamine supplementation doses on infants' cognitive, motor, and language development across the first year. In this double-blind, four-parallel-arm, randomized controlled trial, healthy mothers of exclusively breastfed newborn infants were recruited in Kampong Thom, Cambodia. At 2 weeks postnatal, women (n = 335) were randomized to one of four treatment groups to consume one capsule/day with varying amounts of thiamine for 22 weeks: 0, 1.2, 2.4, and 10 mg. At 2, 12, 24, and 52 weeks of age, infants were assessed with the Mullen Scales of Early Learning (MSEL) and the Caregiver Reported Early Development Instrument (CREDI). Multiple regression and mixed effects modeling suggest that by 6 months of age, the highest maternal thiamine dose (10 mg/day) held significant benefits for infants' language development, but generally not for motor or visual reception development. Despite having achieved standardized scores on the MSEL that approximated U.S. norms by 6 months, infants showed a significant drop relative to these norms in both language domains following trial completion, indicating that nutritional interventions beyond 6 months may be necessary.

Baseline Hemoglobin, Hepcidin, Ferritin, and Total Body Iron Stores are Equally Strong Diagnostic Predictors of a Hemoglobin Response to 12 Weeks of Daily Iron Supplementation in Cambodian Women.

BACKGROUND: The WHO recommends daily iron supplementation for all women in areas where the population-level anemia prevalence is ≥40%, despite the fact that hemoglobin (Hb) concentration is generally considered to be a poor prognostic indicator of iron status. OBJECTIVES: In this secondary analysis, we investigated the predictive power of ten baseline hematological biomarkers towards a 12-week Hb response to iron supplementation. METHODS: Data were obtained from a randomized controlled trial of daily iron supplementation in 407 nonpregnant Cambodian women (18-45 years) who received 60 mg elemental iron as ferrous sulfate for 12 weeks. Ten baseline biomarkers were included: Hb, measured with both a hematology analyzer and a HemoCue; inflammation-adjusted ferritin; soluble transferrin receptor; reticulocyte Hb; hepcidin; mean corpuscular volume; inflammation-adjusted total body iron stores (TBIS); total iron binding capacity; and transferrin saturation. Receiver operating characteristic (ROC) curves from fitted logistic regression models were used to make discrimination comparisons and variable selection methods were used to construct a multibiomarker prognostic model. RESULTS: Only 25% (n = 95/383) of women who completed the trial experienced a 12-week Hb response ≥10 g/L. The strongest univariate predictors of a Hb response were Hb as measured with a hematology analyzer, inflammation-adjusted ferritin, hepcidin, and inflammation-adjusted TBIS (AUCROC = 0.81, 0.83, 0.82, and 0.82, respectively), and the optimal cutoffs to identify women who were likely to experience a Hb response were 117 g/L, 17.3 µg/L, 1.98 nmol/L, and 1.95 mg/kg, respectively. Hb as measured with a hematology analyzer, inflammation-adjusted ferritin, and hepcidin had the best combined predictive ability (AUCROC=0.86). Hb measured with the HemoCue had poor discrimination ability (AUCROC = 0.65). CONCLUSIONS: Baseline Hb as measured with a hematology analyzer was as strong a predictor of Hb response to iron supplementation as inflammation-adjusted ferritin, hepcidin, and inflammation-adjusted TBIS. This is positive given that the WHO currently uses the population-level anemia prevalence to guide recommendations for untargeted iron supplementation.

Low-dose thiamine supplementation of lactating Cambodian mothers improves human milk thiamine concentrations: a randomized controlled trial.

BACKGROUND: Infantile beriberi-related mortality is still common in South and Southeast Asia. Interventions to increase maternal thiamine intakes, and thus human milk thiamine, are warranted; however, the required dose remains unknown. OBJECTIVES: We sought to estimate the dose at which additional maternal intake of oral thiamine no longer meaningfully increased milk thiamine concentrations in infants at 24 wk postpartum, and to investigate the impact of 4 thiamine supplementation doses on milk and blood thiamine status biomarkers. METHODS: In this double-blind, 4-parallel arm randomized controlled dose-response trial, healthy mothers were recruited in Kampong Thom, Cambodia. At 2 wk postpartum, women were randomly assigned to consume 1 capsule, containing 0, 1.2 (estimated average requirement), 2.4, or 10 mg of thiamine daily from 2 through 24 weeks postpartum. Human milk total thiamine concentrations were measured using HPLC. An Emax curve was plotted, which was estimated using a nonlinear least squares model in an intention-to-treat analysis. Linear mixed-effects models were used to test for differences between treatment groups. Maternal and infant blood thiamine biomarkers were also assessed. RESULTS: In total, each of 335 women was randomly assigned to1 of the following thiamine-dose groups: placebo (n = 83), 1.2 mg (n = 86), 2.4 mg (n = 81), and 10 mg (n = 85). The estimated dose required to reach 90% of the maximum average total thiamine concentration in human milk (191 µg/L) is 2.35 (95% CI: 0.58, 7.01) mg/d. The mean ± SD milk thiamine concentrations were significantly higher in all intervention groups (183 ± 91, 190 ± 105, and 206 ± 89 µg/L for 1.2, 2.4, and 10 mg, respectively) compared with the placebo group (153 ± 85 µg/L; P < 0.0001) and did not significantly differ from each other. CONCLUSIONS: A supplemental thiamine dose of 2.35 mg/d was required to achieve a milk total thiamine concentration of 191 µg/L. However, 1.2 mg/d for 22 wk was sufficient to increase milk thiamine concentrations to similar levels achieved by higher supplementation doses (2.4 and 10 mg/d), and comparable to those of healthy mothers in regions without beriberi. This trial was registered at clinicaltrials.gov as NCT03616288.

Can Automated Hematology Analyzers Predict the Presence of a Genetic Hemoglobinopathy? An Analysis of Hematological Biomarkers in Cambodian Women.

Genetic hemoglobinopathies are the most common single-gene disorder worldwide. Some automated hematology analyzers have the capability of flagging individuals who may have hematological disorders based on complete blood count (CBC) biomarkers. We aimed to evaluate the accuracy of a hematology analyzer in identifying genetic hemoglobinopathies in Cambodian women and to determine which hematological biomarkers are the best predictors. A CBC was completed using a Sysmex XN-1000 analyzer and hemoglobinopathies were determined with capillary hemoglobin electrophoresis for 808 nonpregnant Cambodian women. Sysmex XN-1000 Interpretive Program (IP) messages, which flag potential hematological disorders, were produced from CBC results. Then, 2 × 2 tables were used to determine sensitivity and specificity of the IP message "Hemoglobin defect" to detect a genetic hemoglobinopathy. Receiver operating characteristic (ROC) analyses assessed the diagnostic ability of six CBC biomarkers to predict a genetic hemoglobinopathy. In total, 74% of women had a hemoglobinopathy (predominantly Hb E and α-thalassemia). "Hb defect" IP message sensitivity and specificity for genetic hemoglobinopathy detection were 10.4% and 98.6%, respectively. Variable selection strategies yielded a two-variable model including mean corpuscular volume (MCV) and red blood cell (RBC) count (AIC = 99.83, AUCROC = 0.98 (95% CI: 0.97, 0.99)) for the prediction of a homozygous EE disorder. Sensitivity and specificity values do not justify the use of Sysmex XN-1000 IP flag messages for identification of genetic hemoglobinopathies in Cambodian women. Development of an algorithm based on MCV and RBC biomarkers may optimize the screening ability of automated hematology analyzers.

Assessment of salt intake to consider salt as a fortification vehicle for thiamine in Cambodia.

Thiamine deficiency is a public health issue in Cambodia. Thiamine fortification of salt has been proposed; however, the salt intake of lactating women, the target population, is currently unknown. We estimated salt intakes among lactating women (<6 months postpartum) using three methods: repeat observed-weighed intake records and 24-h urinary sodium excretions (n = 104), and household salt disappearance (n = 331). Usual salt intake was estimated by adjusting for intraindividual intakes using the National Cancer Institute method, and a thiamine salt fortification scenario was modeled using a modified estimated average requirement (EAR) cut-point method. Unadjusted salt intake from observed intakes was 9.3 (8.3-10.3) g/day, which was not different from estimated salt intake from urinary sodium excretions, 9.0 (8.4-9.7) g/day (P = 0.3). Estimated salt use from household salt disappearance was 11.3 (10.7-11.9) g/person/day. Usual (adjusted) salt intake from all sources was 7.7 (7.4-8.0) g/day. Assuming no stability losses, a modeled fortification dose of 275 mg thiamine/kg salt could increase thiamine intakes from fortified salt to 2.1 (2.0-2.2) mg/day, with even low salt consumers reaching the EAR of 1.2 mg/day from fortified salt alone. These findings, in conjunction with future sensory and stability research, can inform a potential salt fortification program in Cambodia.

Is untargeted iron supplementation harmful when iron deficiency is not the major cause of anaemia? Study protocol for a double-blind, randomised controlled trial among non-pregnant Cambodian women.

INTRODUCTION: The WHO recommends daily oral iron supplementation for 12 weeks in women and adolescents where anaemia prevalence is greater than 40%. However, if iron deficiency is not a major cause of anaemia, then, at best, untargeted iron supplementation is a waste of resources; at worst, it could cause harm. Further, different forms of iron with varying bioavailability may present greater risks of harm. METHODS AND ANALYSIS: A 12-week three-arm, double-blind, randomised controlled supplementation trial was conducted in Cambodia to determine if there is potential harm associated with untargeted iron supplementation. We will recruit and randomise 480 non-pregnant women (ages 18-45 years) to receive one of three interventions: 60 mg elemental iron as ferrous sulfate (the standard, commonly used form), 18 mg ferrous bisglycinate (a highly bioavailable iron amino acid chelate) or placebo. We will measure ferritin concentrations (to evaluate non-inferiority between the two forms of iron), as well as markers of potential harm in blood and stool (faecal calprotectin, gut pathogen abundance and DNA damage) at baseline and 12 weeks. Mixed-effects generalised linear models will be used to assess the effect of iron on ferritin concentration and markers of potential harm at 12 weeks. ETHICS AND DISSEMINATION: Ethical approval was obtained from the University of British Columbia Clinical Research Ethics Board (H18-02610), the Children's and Women's Health Centre of British Columbia Research Ethics Board (H18-02610) and the National Ethics Committee for Health Research in Cambodia (273-NECHR). Findings will be published in peer-reviewed journals, presented to stakeholders and policymakers globally and shared within participants' communities. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT04017598).

The Homozygous Hemoglobin EE Variant Is Associated with Poorer Riboflavin Status in Cambodian Women of Reproductive Age.

BACKGROUND: Riboflavin is required for erythropoiesis, which is increased in people with hemoglobinopathies due to increased hemolysis and erythrocyte turnover. Dietary intake and status of riboflavin is poor in Cambodia, where hemoglobinopathies are common. OBJECTIVE: We assessed the association between genetic hemoglobin disorders and riboflavin status in women of reproductive age in Cambodia. METHODS: Venous blood samples from 515 Cambodian women of reproductive age, 18-45 y, were analyzed for biomarker status of riboflavin [erythrocyte glutathione reductase activation coefficient (EGRac)], genetic hemoglobin (Hb) disorders, and hematological indices. Linear regression analysis was used to estimate the association between EGRac with Hb, ferritin, and Hb genotypes. EGRac was log transformed in the analyses, and the regression coefficients represent the geometric mean differences. RESULTS: Genetic Hb disorders were present in 57% of the population, with the homozygous hemoglobin E variant (Hb EE) occurring in ∼10% of women (n = 53). Deficient (EGRac ≥1.40) or marginal riboflavin status (EGRac ≥1.30 and <1.40) was observed in 92% (n = 475) of women. The variant Hb EE genotype was associated with 18% (95% CI: 9%, 28%) higher geometric mean EGRac values than the normal Hb AA genotype (P < 0.001). CONCLUSIONS: Although riboflavin biomarker deficiency or marginal status is widely prevalent in Cambodian women, lower riboflavin status was observed more frequently in women with the Hb EE genotype than in women with normal Hb AA. The relation between genetic Hb disorders and riboflavin warrants further investigation. This trial was registered at clinicaltrials.gov as NCT01593423 and NCT02481375.

Macro- and Micronutrients in Milk from Healthy Cambodian Mothers: Status and Interrelations.

BACKGROUND: Except for low thiamin content, little is known about vitamins or macronutrients in milk from Cambodian mothers, and associations among milk nutrients. OBJECTIVES: We measured fat-soluble vitamins (FSVs) and water-soluble vitamins (WSVs), and macronutrients, and explored internutrient associations in milk from Cambodian mothers. METHODS: Milk from women (aged 18-45 y, 3-27 wk postpartum, n = 68) who participated in a thiamin-fortification trial were analyzed for vitamins B-2 (riboflavin, FAD), B-3 (nicotinamide), B-5, B-6 (pyridoxal, pyridoxine), B-7, B-12, A, E [α-tocopherol and γ-tocopherol (γ-TPH)], carotenoids, carbohydrate (CHO), fat, and protein. Milk vitamin B-1 [thiamin, thiamin monophosphate (TMP), thiamin pyrophosphate (TPP)] was previously assessed for fortification effects. Milk nutrient concentrations were compared with the Adequate Intake (AI) values for infants aged 0-6 mo. Pearson correlation was used to examine internutrient associations after excluding nutrients affected by fortification. RESULTS: Fortification increased thiamin and B-1 and decreased γ-TPH. Less than 40% of milk samples met the AIs for all vitamins, and 10 samples did not reach any AI values for the analyzed nutrients. CHO, fat, and energy values were met in 1.5-11.8%, and protein in 48.5%, of the samples. Whereas fat, protein, and energy were related (all r < 0.5; P < 0.001) and associated with FSVs and WSVs, CHO correlated only with some WSVs. TPP was not correlated with B-1 vitamers, but with other WSVs (r = 0.28-0.58; P < 0.019). All FSVs, except α-carotene, were correlated with each other (r = 0.42-0.98; P < 0.002). TPP, FAD, B-2, and B-3 were associated with almost all FSVs (r = 0.24-0.63; P < 0.044). CONCLUSIONS: Cambodian women might not provide sufficient nutrients to their exclusively breastfeeding infants. Besides thiamin, all other vitamins measured were much lower than the AI. There were many strong correlations among macronutrients and vitamins; the extent to which these are explained by maternal diet, milk volume, maternal physiology, or genetics requires additional exploration.

Thiamine dose response in human milk with supplementation among lactating women in Cambodia: study protocol for a double-blind, four-parallel arm randomised controlled trial.

INTRODUCTION: Thiamine (vitamin B1) deficiency remains a concern in Cambodia where women with low thiamine intake produce thiamine-poor milk, putting their breastfed infants at risk of impaired cognitive development and potentially fatal infantile beriberi. Thiamine fortification of salt is a potentially low-cost, passive means of combating thiamine deficiency; however, both the dose of thiamine required to optimise milk thiamine concentrations as well as usual salt intake of lactating women are unknown. METHODS AND ANALYSIS: In this community-based randomised controlled trial, 320 lactating women from Kampong Thom, Cambodia will be randomised to one of four groups to consume one capsule daily containing 0, 1.2, 2.4 or 10 mg thiamine as thiamine hydrochloride, between 2 and 24 weeks postnatal. The primary objective is to estimate the dose where additional maternal intake of thiamine no longer meaningfully increases infant thiamine diphosphate concentrations 24 weeks postnatally. At 2, 12 and 24 weeks, we will collect sociodemographic, nutrition and health information, a battery of cognitive assessments, maternal (2 and 24 weeks) and infant (24 weeks only) venous blood samples (biomarkers: ThDP and transketolase activity) and human milk samples (also at 4 weeks; biomarker: milk thiamine concentrations). All participants and their families will consume study-provided salt ad libitum throughout the trial, and we will measure salt disappearance each fortnight. Repeat weighed salt intakes and urinary sodium concentrations will be measured among a subset of 100 participants. Parameters of Emax dose-response curves will be estimated using non-linear least squares models with both 'intention to treat' and a secondary 'per-protocol' (capsule compliance ≥80%) analyses. ETHICS AND DISSEMINATION: Ethical approval was obtained in Cambodia (National Ethics Committee for Health Research 112/250NECHR), Canada (Mount Saint Vincent University Research Ethics Board 2017-141) and the USA (University of Oregon Institutional Review Board 07052018.008). Results will be shared with participants' communities, as well as relevant government and scientific stakeholders via presentations, academic manuscripts and consultations. TRIAL REGISTRATION NUMBER: NCT03616288.

Including 60 mg Elemental Iron in a Multiple Micronutrient Supplement Blunts the Increase in Serum Zinc after 12 Weeks of Daily Supplementation in Predominantly Anemic, Nonpregnant Cambodian Women of Reproductive Age.

BACKGROUND: Multiple micronutrient (MMN) supplementation may result in interaction effects due to competing absorptive pathways of trace elements. OBJECTIVES: The aim of this study was to investigate the effect of MMN supplementation with or without iron on serum zinc, selenium, and copper concentrations in Cambodian women. METHODS: In a 2 × 2 factorial double-blind randomized 12-wk trial, predominantly anemic, nonpregnant women (aged 18-45 y) received daily 60 mg of iron (Fe; n = 201); 14 other micronutrients including zinc (15 mg), selenium (65 µg), and copper (2 mg), but no iron (MMN; n = 202); 60 mg iron plus MMN (Fe + MMN; n = 206); or a placebo (n = 200). Fasting morning blood was collected at baseline and 12 wk from women in 26 villages in Kampong Chhnang province. Serum zinc, selenium, and copper concentrations (secondary outcomes of the randomized controlled trial) were measured using inductively coupled plasma mass spectrometry. Generalized linear regression was used to estimate intervention effects [ß coefficient (95% CI)] for Fe (with or without MMN) and MMN (with or without Fe) after testing for the presence of an Fe × MMN interaction. RESULTS: A total of 760 women completed the trial. Zinc deficiency prevalence at baseline was 45% (inflammation-adjusted serum zinc <10.7 µmol/L). A significant Fe × MMN interaction (P = 0.02) was detected in the 2 × 2 analysis with serum zinc concentration as the outcome: the MMN group had a higher mean serum zinc concentration at 12 wk (12.3 µmol/L; 95% CI: 12.2, 12.4 µmol/L) compared with all other groups, and the Fe + MMN group had a higher mean serum zinc concentration (11.6 µmol/L; 95% CI: 11.5, 11.7 µmol/L) compared with the Fe group (11.0 µmol/L; 95% CI: 10.9, 11.0 µmol/L) and the placebo group (11.2 µmol/L; 95% CI: 11.1, 11.4 µmol/L). CONCLUSIONS: The inclusion of 60 mg iron in the daily MMN formulation may be interfering with the absorption and/or metabolism of supplemental zinc in Cambodian women. This is of particular concern when MMN supplementation is implemented in populations with risk of zinc deficiency. This trial was registered at clinicaltrials.gov as NCT-02481375.

The Effect of Daily Iron Supplementation with 60 mg Ferrous Sulfate for 12 Weeks on Non-Transferrin Bound Iron Concentrations in Women with a High Prevalence of Hemoglobinopathies.

There is a lack of evidence for the safety of untargeted daily iron supplementation in women, especially in countries such as Cambodia, where both anemia and hemoglobinopathies are common. Our aim was to assess serum non-transferrin bound iron (NTBI), a toxic biochemical that accumulates in blood when too much iron is absorbed, in Cambodian women who received daily iron supplements in accordance with the 2016 global World Health Organization (WHO) guidelines. We used fasting venous blood samples that were collected in a 2015 supplementation trial among predominantly anemic Cambodian women (18⁻45 years). Serum NTBI was measured with use of the FeROS™ eLPI assay (Aferrix Ltd., Tel-Aviv, Israel) in randomly selected sub-groups of women who received 60 mg daily elemental iron as ferrous sulfate (n = 50) or a placebo (n = 50) for 12 weeks. Overall, n = 17/100 (17%) of women had an elevated serum NTBI concentration (≥0.1 µmol/L) at 12 weeks; n = 9 in the Fe group and n = 8 in the placebo group. Elevated serum NTBI concentration was not associated with age, iron supplementation, transferrin saturation or severe hemoglobinopathies (p > 0.05). In this population of women with a high prevalence of hemoglobinopathies, we found that daily iron supplementation was not associated with elevated serum NTBI concentrations at 12 weeks, as compared to placebo.

Comparison of Human Milk Fatty Acid Composition of Women From Cambodia and Australia.

BACKGROUND: Human milk is a rich source of omega-3 long-chain polyunsaturated fatty acids, which are postulated to be important for brain development. There is a lack of data on the human milk fatty acid composition of Cambodian women compared with data from Western women. Research Aim: The aim of this study was to determine the human milk fatty acid composition of women living in Cambodia and compare it with that of women living in Australia. METHOD: Human milk samples from Cambodian ( n = 67) and Australian ( n = 200) mothers were collected at 3 to 4 months postpartum. Fatty acid composition was analyzed using capillary gas chromatography followed by Folch extraction with chloroform/methanol (2:1 v/v), and fat content was measured gravimetrically. RESULT: Compared with Australian participants, human milk from Cambodian participants contained a significantly lower level of total fat (2.90 vs. 3.45 g/dL, p = .028), lower percentages of linoleic acid (9.30% vs. 10.66%, p < .0001) and α-linolenic acid (0.42% vs. 0.95%, p < .0001), but higher percentages of arachidonic acid (0.68% vs. 0.38%, p < .0001) and docosahexaenoic acid (0.40% vs. 0.23%, p < .0001). CONCLUSION: Differences in human milk fatty acid composition between Cambodian and Australian participants may be explained by differences in the dietary patterns between the two populations.

Concentrations of Water-Soluble Forms of Choline in Human Milk from Lactating Women in Canada and Cambodia.

Choline has critical roles during periods of rapid growth and development, such as infancy. In human milk, choline is mostly present in water-soluble forms (free choline, phosphocholine, and glycerophosphocholine). It is thought that milk choline concentration is influenced by maternal choline intake, and the richest food sources for choline are of animal origin. Scarce information exists on milk choline from countries differing in animal-source food availability. In this secondary analysis of samples from previous trials, the concentrations of the water-soluble forms of choline were quantified by liquid chromatography-tandem mass spectrometry in mature milk samples collected from lactating women in Canada (n = 301) and in Cambodia (n = 67). None of the water-soluble forms of choline concentrations in milk differed between Canada and Cambodia. For all milk samples (n = 368), free choline, phosphocholine, glycerophosphocholine, and the sum of water-soluble forms of choline concentrations in milk were (mean (95%CI)) 151 (141, 160, 540 (519, 562), 411 (396, 427), and 1102 (1072, 1133) µmol/L, respectively. Theoretically, only 19% of infants would meet the current Adequate Intake (AI) for choline. Our findings suggest that the concentrations in milk of water-soluble forms of choline are similar in Canada and Cambodia, and that the concentration used to set the infant AI might be inaccurate.

Experiences and lessons learned for delivery of micronutrient powders interventions.

An effective delivery strategy coupled with relevant social and behaviour change communication (SBCC) have been identified as central to the implementation of micronutrient powders (MNP) interventions, but there has been limited documentation of what works. Under the auspices of "The Micronutrient Powders Consultation: Lessons Learned for Operational Guidance," three working groups were formed to summarize experiences and lessons across countries regarding MNP interventions for young children. This paper focuses on programmatic experiences related to MNP delivery (models, platforms, and channels), SBCC, and training. Methods included a review of published and grey literature, interviews with key informants, and deliberations throughout the consultation process. We found that most countries distributed MNP free of charge via the health sector, although distribution through other platforms and using subsidized fee for product or mixed payment models have also been used. Community-based distribution channels have generally shown higher coverage and when part of an infant and young child feeding approach, may provide additional benefit given their complementarity. SBCC for MNP has worked best when focused on meeting the MNP behavioural objectives (appropriate use, intake adherence, and related infant and young child feeding behaviours). Programmers have learned that reincorporating SBCC and training throughout the intervention life cycle has allowed for much needed adaptations. Diverse experiences delivering MNP exist, and although no one-size-fits-all approach emerged, well-established delivery platforms, community involvement, and SBCC-centred designs tended to have more success. Much still needs to be learned on MNP delivery, and we propose a set of implementation research questions that require further investigation.

Comparable Performance Characteristics of Plasma Thiamine and Erythrocyte Thiamine Diphosphate in Response to Thiamine Fortification in Rural Cambodian Women.

Background: Traditionally, vitamin B1 status is assessed by a functional test measuring erythrocyte transketolase (ETK) activity or direct measurement of erythrocyte thiamine diphosphate (eThDP) concentration. However, such analyses are logistically challenging, and do not allow assessment of vitamin B1 status in plasma/serum samples stored in biobanks. Using a multiplex assay, we evaluated plasma concentrations of thiamine and thiamine monophosphate (TMP), as alternative, convenient measures of vitamin B1 status. Methods: We investigated the relationships between the established biomarker eThDP and plasma concentrations of thiamine and TMP, and compared the response of these thiamine forms to thiamine fortification using samples from 196 healthy Cambodian women (aged 18-45 years.). eThDP was measured by high performance liquid chromatography with fluorescence detection (HPLC-FLD) and plasma thiamine and TMP by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Plasma thiamine and TMP correlated significantly with eThDP at baseline and study-end (p < 0.05). Among the fortification groups, the strongest response was observed for plasma thiamine (increased by 266%), while increases in plasma TMP (60%) and eThDP (53%) were comparable. Conclusions: Plasma thiamine and TMP correlated positively with eThDP, and all thiamine forms responded significantly to thiamine intervention. Measuring plasma concentrations of thiamine forms is advantageous due to convenient sample handling and capacity to develop low volume, high-throughput, multiplex assays.