Problem statement
The recommended iron densities of complementary foods (CF) for infants 12-23 months are 1.6 and 0.8mg/100kcal assuming low and average bioavailability, for 6-11 months these densities rank invariably higher (4.6-7.7mg/100kcal and 2.4-4mg/100kcal, respectively) [54]. The iron provided in IG1 accounted for an iron density of 0.86mg iron per 100kcal, and for IG2/IG3 2.33mg iron per 100kcal, respectively. Iron and zinc are considered to be the limiting nutrients in family nutrition in developing countries. Recommended intakes of iron and to a lesser extent of zinc are still unlikely to be provided by improved traditional CF in the first year of life, when portion size is limited and iron gap is biggest. During a child´s first year of life, growth and therewith blood creation is particular high in the child, so are iron needs [55]; [56]. The special iron needs of children under age two get palpable by the increased prevalence of anemia among children aged 6 to 23 months as elaborated by Kotecha based on the data of the Indian NFHS-3, and similarly confirmed by baseline data of the current trial [24]. Kotecha suggests low-birth weights infants, premature infants, and infants of mothers with anemia to require additional iron from two months of age on, otherwise iron stores remain insufficient and iron demands for rapid growth are not met [57]. Ruel et al. concluded that the densities for iron and zinc in traditional complementary recipes in resource-constraint environments remain highly inadequate for children younger than one year, even after inclusion of locally available fortified corn-soy blend (CSB) and optimal combination of locally available, acceptable and affordable foods. An overall challenge remains for targeted beneficiaries to meet appropriate infant feeding practices by adequate nutrient density, consistency and texture, as well as frequency of feeding [58].
In the current study mothers were informed that the feeding program has a supplementary purpose to enhance child´s nutrient intake, still at endline assessment of 189 mothers the majority (74.1%) reported that the meal offered at the nutrition program replaced a meal at home, with one mother stating that only due to the rare attendance of her child (due to disliking the ALP/MLP taste) the meal did not replace a meal at home, and merely 25.4% indicated that the meal supplemented the dietary routine of the child. The reason for meal replacement (n=137), was “the time of nutrition program was the same time when child would receive food at home anyway” (89.8%), thus “children had full stomach after program, could not eat extra food at home” (2.2%), or “because other family members can eat more at home, if one child eats outside” (8.0%). The hosting of the nutrition program in the late afternoon was intentionally chosen, in order that caretakers may be free after work to bring their child to the nutrition program. Still, observations showed children were frequently accompanied by older siblings or even came alone to the program. Altogether the practice of replacing a meal at home is likely to have diminished the possible effect of the low-dose feeding trial. Still, the locally produced supplementary meals were composed of affordable and available ingredients, thus constitute a sustainable complementary food option to be adapted by caregivers after closure of the feeding program. Accordingly, at endline assessment of 188 mothers, more than every fourth (28.2%) indicated that they have modified their cooking behavior at home -inspired by the nutrition program. Of these 53 mothers, 32.1% improved their hygienic behavior (proper cleaning of utensils, intensified washing of vegetables, using food cover, serving spoon), and 83.0% changed composition of their diet (add more vegetables/leafy vegetables, started to use or to add more Amaranthus/Moringa leaves, use more frequently egg or soybean). 157 mothers indicated reasons for not having changed anything or problems faced when trying to change something (no money (70.1%), husband did not like a change (40.1%), mother felt unable to change something (8.3%), time constraints (1.9%), and 55.5% of cases stated they felt no need to change anything e.g. as home food is perceived to be of very good quality). To conclude economic as well as social limitations stand out as main reasons; combined with lack of awareness of the need for modifications.
The Copenhagen Consensus 2008 concluded that nutrition-focused interventions (micronutrient supplementation, fortification as well as community-based nutrition promotion) generate highest economic returns [59], thus are of priority when advancing the well-being of people in developing countries.
The WHO recommends a daily dose of 10-12.5mg elemental iron amongst infants aged 6 to 23 months when the prevalence of anemia is 40% or higher [60]. The absolute absorption efficiency depends on intestinal factors (host-related factors), individual´s iron status or infection state (systemic factors), the dose and form of iron compounds and absorption modifiers consumed with the food vehicle (diet-related factors) [34].
Food synergies in diversified study meals
Food synergy is considered as a key to healthy diet with food components deemed „to act in concert on health“ implying additive effects, this means the whole is more than the sum of its parts. Food consists of a biological complexity developed under evolutionary control, that cannot be imitated by single nutrients. The focus on isolated nutrients in nutrition research may be counterproductive and less-beneficial [61], thus this study investigates the effect of diversified meals only (IG1), and either enriched with whole-food leaf powder (IG2) or an adjusted amount of industrially synthesized isolated micronutrients (IG3). Between IG2 and IG3 no significant differences in Hb concentrations at different assessment points were observed (Table 6, Table 7).
The bioavailability of iron is increased by small amounts of animal-protein “meat factor”, or by ascorbic acid [62]. The latter however may experience substantial losses during storage or preparation, in particular in the presence of oxygen, metals, humidity and/or high temperatures if the food is not appropriately packaged or the vitamin C not encapsulated [63].
Proteins from other food sources in particular eggs, milk and dairy products (especially casein) inhibit iron absorption. Serum ferritin was reported to be positively associated with flesh foods intake and negatively related to dairy product consumption [34]. Still, in the current trial iron absorption may be equally impaired by the egg or milk proteins across all study groups, as the only variation is the ALP/MLP (IG2) or adjusted amount of TopNutri (IG3).
Zinc and iron were found to show competitive interactions, similarly calcium may have an acute inhibitory effect on iron absorption [34]. The adverse effect is dependent on the calcium amount administered rather than the Ca:Fe molar ratio [64]. Negative effects have been shown with the addition of 165mg calcium from milk products [65]. The maximum inhibitory effect is suggested to be achieved at calcium doses of 300mg (77% inhibition) in a meal, but already starts at 40mg (39% inhibition) [64]. Still, reported negative effects of calcium on iron absorption are predominantly assessed in single-meal studies; whereas multiple-meal studies -embracing a huge amount of other inhibitors and promoters, suggest calcium to have merely a limited effect [65]. Moreover it remains uncertain if the observed adverse effects are due to the increased calcium or rather related to inhibitory milk proteins of dairy products commonly applied as primary source of calcium in single-meal studies [64]. The calcium content per 100g Halwa including half egg/Khechuri was highest for IG3 (148.1mg/117.4mg), followed by IG2 (119.8mg/88.8mg), with IG1 providing lowest calcium amounts (70.8mg/40.1mg). Thus, a potential inhibitory effect of calcium on iron absorption is conceivable with the decrease in absorption rate to be considered highest for IG3.
Measuring the molar ratio of phytate/minerals in the diet predicts bioavailability of minerals in the body. Phytate (PA):mineral molar ratios (PA:Fe, PA:Zn, PA:Ca, Ca*PA/Zn, Ca:P, Oxalate:Ca, Zn:Fe) were calculated based on the Indian Food Composition [66] and Annex 1 for the study recipes (results not shown). Hereby, for PA:Fe the critical ratio of 1:1 -as suggested by Hurrel for plain cereal or legume-based meals [67], has been exceeded by all study groups. For an exemplified serving size of 100g, the highest PA:Fe ratio was assessed for diversified study meals only (IG1, PA:Fe ratio: 3.97, 2.96), followed by diversified meals plus ALP/MLP (IG2, PA:Fe ratio: 1.41, 1.35), with diversified meals plus an adjusted amount of TopNutri (IG3, PA:Fe ratio: 1.37, 1.31) having the lowest ratio for Halwa including half egg/Khechuri, respectively. Moreover for IG3 the Ca*PA/Zn ratio highly exceeded the critical value of 0.5, -as suggested by Ferguson [68], with 8.98 and 9.47 for Halwa including half egg and Khechuri, respectively. The latter indicates that calcium may influence zinc absorption for IG3.
Foods considered as being high in PA are defined to contain >400mg PA/100g food [69]. Yet, the dose-dependent inhibitory effect of phytate on iron absorption is reported to start at low concentrations of 10mg/meal [70]. The PA contents of all study meals were <65mg/100g serving size of Halwa/Khechuri. According to Hurrel [67], [71] for composite meals, containing ascorbic acid from vegetables or meat as enhancers, a PA:Fe ratio of 6:1 may be acceptable. Thus, the PA content in the current study meals may not be a major inhibitory factor.
The benefits and limitations of food-based approaches for combating iron-deficiency and malnutrition by dietary diversification are discussed in detail by reports of the FAO [15], [72]. Evaluations of the efficacy of multi-sectoral approaches for dietary diversification is challenging and remains inconclusive, due to the complexity of holistic food-based strategies, the variety of nutritional components contained in food, large number of confounders and difficulties for study design in regard to short-term and long-term effects. Food-based approaches for dietary diversification focus on food and livelihood-based models (e.g. home gardening and crop-diversification, the increased availability and consumption of animal-sourced foods, education and training, access to sanitary facilities, social services and health care), thereby strive for prevention and sustainability.
In the current trial altogether diversified meals alone (IG1) were found to have most effectively increased the Hb concentrations as opposed to the CG at 2nd, 3rd, and endline assessment. Leaf-powder enriched meals (IG2), or meals with an adjusted amount of MNP showed no superior effect toward the CG. The superiority of IG1 vs. CG in regard to Hb concentrations was also affirmed by the conservative Bonferroni-post-hoc test at 3rd assessment (subtext Table 6, Table 7). Concerning growth indices the effect of IG1 appears less conclusive. At 3rd assessment IG1 showed superiority toward CG concerning WAZ scores. Moreover IG1 showed highest mean time investment in caring activities, and at home mothers were sitting next to their child during eating on significant more days during a five-week assessment as compared to the other study groups (Table 2). This increased caring may promote increased food ingestion in the child, thus may positively contribute to anemia reduction or growth development in this study.
Leaf powder and bioavailability studies
Moringa oleifera is especially promoted due to its satisfying iron content. Gallaher et al [73] investigated the iron bioavailability of dried Moringa leaf powder (MLP) (0.5% dry weight basis diet) in rats, and came to the conclusion that the high phytic acid content of the leaves implies not only a low bioavailability of iron from the Moringa supplement itself but also inhibits the absorption of iron provided from the general diet. Rats receiving commercial animal feed and MLP at 10%, experienced significant lower increases in Hb and weight as compared to another experimental group being fed MLP at 5% [74].
Equally, researches of the University of Pretoria [75], [76] concluded that the in vitro iron and zinc bioaccessibility of adding MLP in the weight amount of 5% to a standardized pearl millet porridge (dry weight basis) was superior to the adding of 15% of Moringa with regard to the fulfillment of physiological reference intake levels. As compared to other dried food-to-food fortifications e.g. hibiscus leaves, baobab fruit pulp, mango-carrot premix; MLP resulted in the lowest increases of iron and zinc accessibility attributable to the high calcium, phenolic, phytic and organic acid content. MLP increased (due to its high iron content) but also decreased bioaccessibilies, altogether a negative impact on the bioaccessibility of the porridge itself has to be considered due to the high levels of iron and zinc inhibitors. All studies used a realistic single-meal food matrix, which suggests that there is no amplified impact on iron absorption as it may be in studies utilizing isolated plant products. Still, it has to be noted that studies on single-meals versa whole diets tend to exaggerate the effect of nutrient interactions involving iron absorption [34]. In the current trial the applied overall leaf powder dose of ALP/MLP was rather low accounting for 3% in total (wet weight basis of overall served study meal), and in regard to MLP 1%. Without cooking water and not considering the served egg, fortification levels rank at maximum double. Still, a theoretical low dosage of <1%-5% MLP could exceed the critical amount according to investigations by Gallaher et al [73], however still may be an enrichment according to considerations of Merwe et al. [75] as outlined above. Tessera et al. [77] reported the bioavailability of iron to be highest for 5% MLP blending while zinc and calcium were found to be well available in blending up to 20% MLP with wheat flour on the basis of molar ratios of minerals and antinutrients.
According to analysis by the LA Chemie University of Hohenheim the phytate contents in dried ALP/MLP were <0.05%, thus not exactly detectable. According to the Indian Food Composition Table [66] fresh Amaranthus leaves contain less phytic acid than fresh Moringa leaves e.g. (4.89mg/100g vs. 128mg/100g). Other analysis published revealed phytic acid contents accounting for 234mg in 100g of Amaranthus leaf powder [78]; or ranging from 0.265mg [18] to 2100mg [79] in 100g of dried Moringa leaves (Supplementary Material 1).
In the current trial the mean adjusted Hb concentrations assessed in IG2 showed no significant differences toward the CG across all assessment points (Table 6, Table 7). Moreover IG2 was slightly worse than the CG and significantly inferior to IG1 concerning Hb concentrations at 2nd assessment (Table 6) or in regard to the Hb changes occurred between baseline and 2nd assessment (Table 7), possibly attributable to antinutrients (phytic acid, polyphenols, oxalate) or nutrient-to-nutrient interactions. Still, at the 3rd and endline assessment IG2 remained comparable to all intervention groups.
Similarly, in regard to adjusted means of growth indices (HAZ, WAZ, WHZ); IG2 failed to show significant differences as opposed to the CG. Concerning adjusted HAZ scores IG2 was significantly inferior to IG3 at endline, and showed most poor changes in scores over time amongst all study groups (Figure 7). For adjusted WAZ-scores IG2 was found significantly inferior to IG3 at 3rd assessment. In regard to adjusted WHZ scores at endline the highest scores were found for IG2 (lowest WHZ prevalence), moreover IG2 showed significant better WHZ scores than IG3 at endline, also confirmed by the conservative Bonferroni-post-hoc test (subtext, Supplementary Material 10). It has to be noted that IG3 experienced excessive faltering in WHZ scores at endline, but showed the best performance at 3rd assessment (Figure 9). The positive effect on reduction of wasting prevalence may be attributable to the holistic amino acid composition present in ALP/MLP (IG2) (Supplementary Material 2), and TopNutri (IG3) equally containing high quality protein (amino acid score of 100 [80]).
IG2 showed lowest consumption rates throughout the trial as compared to the other intervention groups, significantly inferior to IG1 during IP1 and IP3. Similarly mothers were significantly less sitting next to their child during eating at home, in IG2 versa IG1 (Table 2). Altogether the lower consumption rates during the program and less attentive eating athmosphere at home, may have negatively affected the reduction in anemia rates or growth development.
Acceptability of MLP and left-over rate of distributed portions
The feasibility and acceptability of using dried Moringa leaves as nutrition supplement was rated to be high by several studies. In India, a pilot study on children aged 1-5 years over one month found the adding of 5-7g leaf powder /100g food as well accepted. A maximum left-over of 30% of distributed portion size was reported, equally observed in the control group consuming regular recipes [81]. Similarly Boateng et al. 2017 reported the daily MLP-supplementation of 5g MLP sprinkled over usual foods or incorporated in a 35g cereal-legume blend (15% fortification, dry weight) to be well-accepted in Ghanian infants aged 8-12 months, with a daily left-over rate of 13.8% or 28.5%, respectively during a 14-day period [82]. Boateng et al. [83] reviewed common MLP fortification levels applied in complementary food blends ranging from 1 to 25 %, with the authors´ recommendation of acceptable MLP fortification levels being 1 to 15 % (the minimum amount of MLP to significantly improve nutritional value was estimated with 10%). In the current trial the general acceptability of ALP/MLP application was perceived as good among children (as no child was recorded to reject the food -probably due to the young age). Few caretakers however seemed more critical towards the greenish color and uncommon “leafy” taste. Moreover the belief/fear that Moringa may decrease the blood pressure in a harmful way guides the skepticism amongst Adivasis (as being named in the baseline survey by 10 out of 43 caretakers as reason for not consuming Moringa at home). The lowest rate of consumption was assessed for IG2, possibly an indirect consequence of caretaker´s influence on the feeding process or directly on child´s appetite due to discomfort in taste. Few pregnant women (also entitled to participate in the nutrition program) mentioned to suffer from nausea when smelling the ALP/MLP-enriched meal.
Nutrition intervention studies including the feeding of green leafy vegetables (GLV) amongst others different Amaranthus species
Studies on the consumption of GLV are diverse in their results. Nawiri et al. [84] provided 80g fresh/re-hydrated sun-dried cowpea and Amaranthus tricolor leaves to Kenian pre-school children over a period of 13 weeks (IG1/IG2, n=56/20, baseline Hb 11.5/11.6g/dl), versa fresh/re-hydrated sun-dried white cabbage (CG1/CG2, n=51/25, baseline Hb 12.4/12.3g/dl). A non-significant but positive intra-group pre-post increase for Hb concentrations in the IG (0.7/0.6g/dl) was reported, with CG showing no increases (-0.1/-0.1g/dl), (descriptive study analysis). Similarly Egbi et al. [85] (inferential study) supplemented Ghanian children aged 4 to 9 years (IG, n=53, baseline Hb 11.8g/dl; CG, n=51, baseline Hb 11.7g/dl) with beans/tomato stews and groundnut soup (CG) enriched with dried composite GLV powder including Amaranthus cruentes (IG) five times a week over a period of three months, and found a significant higher increase in Hb (0.43g/dl versa -0.35g/dl) as well as higher decrease in anemia prevalence (Hb<11.5g/dl) from 41.5% to 33.3% in IG versa 37.3% to 57.5% in CG, respectively.
Two other inferential studies however could not show a positive effect of GLV on Hb: Hoeven et al. [86] found no significant differences in Hb increases between IG (n=86, Hb baseline 12.8g/dl, Hb increase 0.3g/dl) and CG (n=81, Hb baseline 12.7g/dl, Hb increase 0.4g/dl) in school-children aged 6-12 years receiving a starchy school meal including either 300g cooked leafy vegetables with Amaranthus cruentes accounting for 80-100% of GLV, versa only small amounts of cabbage or a teaspoon legumes or meat/soya mince over a period of 62 school days. The authors conclude that the regular consumption of GLV showed no effect on micronutrient status (Hb, serum ferritin, serum zinc and serum retinol) in South African children with mild deficiencies. Pee et al. [87] investigated potential differences in the provision of dietary sources rich in carotene/retinol. Hereby either meals predominantly based on GLV including three Amaranthus types (IG1, n=45, baseline Hb 11.1g/dl) versa meals based on carotene-rich fruits (IG2, n=49, baseline Hb 11.1g/dl), retinol-rich foods (IG3, n=48, baseline Hb 11.1g/dl) or foods low in carotene or retinol (CG, n=46, baseline Hb 11.2g/dl) were fed to anemic school-children with two meals a day over a period of nine weeks in Indonesia. The subjects fed on GLV experienced the smallest increase in Hb (0.1g/dl) amongst all four study groups, which was significant lower as compared to IG2 (0.5g/dl) and IG3 (0.5g/dl) possibly attributable to the low bioavailability of non-haem iron.
The effectiveness of leaf concentrates in improving the human nutritional status has been reviewed by Davys et al. [72] relating to studies in the more distant past. Positive effects on height and weight development, Hb concentration, vitamin A status, and diminished morbidity were observed as opposed to control groups, however the statistical relevance has not been discussed in detail.
Nutrition intervention studies including the feeding of Moringa oleifera
Indigenous nutrient-rich plant sources like Moringa oleifera are increasingly discussed as potential sustainable solution to combat malnutrition in children [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102]. Descriptive studies (intra-group comparisons) comment positively on MLP application as preventive or treatment approach however bear distinct limitations: Isingoma et al [99] reported the application of a fermented millet porridge with 7% MLP (IG, n=26, baseline Hb 9.6g/dl) to be comparable to F-100 (CG, n=25, baseline Hb 9.8g/dl) in the rehabilitation of SAM children aged 7 to 36 months, Uganda. By reaching discharge criteria after 5 to 7 days children achieved significant increases in Hb of 0.6g/dl (IG) versa 0.2g/dl (CG), respectively. The wasting z-scores similarly improved by a comparable rate of 1.4 versa 0.9 within the study groups. Srikanth et al [100] successfully treated Indian children suffering from protein energy malnutrition grade (PEM) I or II (aged < 5 years), with the application of 30g MLP added to child´s daily diet (n=30) over a study duration of two months. There was a trend of higher weight gains for children of the IG as compared to the CG (n=30), however no report on compliance (mothers were in charge to administer the MLP to their children), nor a description of daily diet in the CG. Asante et al [102] reported an Hb increase of 0.9g/dl (baseline Hb 9.3 g/dl) after application of 15g MLP (IG, n=25) over a study period of four weeks, however neither baseline Hb concentrations nor increases observed in the CG (F-100, n=10) were presented. During the rehabilitation of Ghanian children (aged 6 to 36 months and suffering from PEM), there was a clear trend of higher weight increases for IG, with the authors of the study concluding MLP to have the potential to significantly contribute to the management of malnutrition. Nnam et al [101] conducted a study in Nigeria on children aged 6 to 12 months (unclear inclusion/exclusion criteria, baseline Hb 10.7g/dl in IG/CG, respectively) over a period of four weeks. Children in the IG (n=20) received a fermented maize porridge with MLP (3.67mg iron/day), with the CG (n=20) receiving the same porridge without MLP. Merely for the IG significant increases in Hb (2.3g/dl) as well as serum ferritin (26.2µg/l) could be observed, with zero increase in CG, respectively.
Two inferential studies (inter-group comparisons) commented positively on the effects of MLP application on Hb: Andrew [88] fed a with 25g MLP enriched maize porridge (IG, n=64, baseline Hb 7.4g/dl) to SAM children aged 6 to 24 months attending nutrition rehabilitation in Tanzania over a period of three months. A significant higher Hb increase was observed for IG (4.1g/dl) as opposed to CG (0.2g/dl) (n=76, baseline Hb 7.1g/dl). Similarly gains in weight, MUAC (cm), WAZ and WHZ were significantly higher in IG versa CG. Moreover a reduction on diarrhea prevalence from 13% at baseline to 1.4% at endline assessment was reported for IG.
Saturnin et al. [94] provided 10g MLP/d (IG, n=44, baseline Hb 10.3g/dl) to moderately acute malnourished (MAM) children which was applied to usual diet of the 6 to 30 months old children in Benin. A significant higher increase in Hb of 1.6g/dl was assessed for IG as compared to CG (-0.6g/dl) (n=40, baseline Hb 10.4g/dl) after six months.
A range of other inferential studies showed no significant increases in Hb in IG versa CG: in a study in Burkina Faso conducted by Zongo et al [98] the addition of 10g MLP/d to standard care diet (IG, n=52, baseline Hb 9.7g/dl) was efficient to increase weight gains but not Hb concentrations (-0.1g/dl (IG) versa ‑1.0g/dl (CG)) when compared to CG (n=58, baseline Hb 10.5g/dl). Hereby the admission period for SAM children aged 6 to 59 months accounted for an average of 36 days (IG) and 57 days (CG) due to significant higher weight gains in IG (8.9g/kg/d) versa CG (5.7 g/kg/d), respectively. Another trial by Zongo et al [97] conducted in Burkina Faso on pre-school children aged 12 to 59 months was ineffective in changing the nutritional status (HAZ, WHZ) by applying 30g MLP over the day for 12 weeks (IG n=60, CG n=59). A study on the Philippines conducted by Serafico et al. [96] found no significant effects of snack foods enriched with 3g MLP on Hb, weight or height after 120 days in 8 to 10 year old underweight school children (mean baseline Hb of 12.5g/dl for IG n=61/CG n=60, respectively). Similarly, in a trial by Menasria et al [95] (assigning its subjects during analysis in the categories “actual intervention received” and “non-compliers”, thus not following the intention-to-treat approach), the application of 16g MLP over six months showed no significant effects on Hb, Ferritin, HAZ or WHZ in Cambodian children aged 6 to 23 months as opposed to the CG (baseline Hb 10.1g/dl, n=39 (IG) versa 9.9g/dl, n=79 (CG)). Hb increases accounted for 0.5g/dl (IG) and 0.4g/dl (CG). Equally a study by Glover-Amengor et al [93] found no significant increase in Hb in Ghanian school children aged 5 to 12 years by applying 2.4g up to 5.1g MLP (depending on body weight) along with study meals three times a week over nine weeks as compared to CG (baseline Hb 10.6g/dl for IG n=69/CG n=76, respectively). Another trial conducted in Ghana by Boateng et al [92] failed to show significant effects of a daily dose of 5g MLP –either as part of cereal-legume blend (IG1, n=80) or as a supplement sprinkled on infant´s usual home-based diet (IG2, n=74), on Hb concentrations or growth indicators of children aged 8 to 12 months after a total trial period of 16 weeks (baseline Hb n.a.) as compared to CG (n=83) receiving the cereal-legume blend without enrichments. Also Perlas et al [90] could not proof any significant increases in Hb, serum ferritin, weight or height as compared to the CG, after providing snacks enriched with 3g MLP on a daily basis over 120 days to Filipino school children (WAZ<-2SD) aged eight to ten years (baseline Hb 12.5g/dl for IG n=61/CG n=60, respectively). Lonati et al [91] concluded the application of 3 to 5g MLP (IG, n=88) to have no significant effects on the prevalence of anemia (Hb<11.5g/dl) in Cameroon school children aged 5 to 8 years as compared to placebo meal without MLP (CG, n=95). Merely the increase in height/HAZ was reported as significantly higher for IG than CG after 18 weeks.
Yet, there is no general agreement on an outstanding nutritional benefit of local plant sources in terms of preventing or treating malnutrition. Due to the inconsistency in the quality of human intervention studies it is difficult to come to a conclusive judgment.
Studies including supplementation of TopNutri and other micronutrient supplements
The bioavailability of nutrients from supplements/fortificants when taken along with a meal is affected by the same factors as those present in food, with the net effect depending on the ratio of promoters and inhibitors [34]. Among reviewed complementary food supplements by Pee et al. TopNutri provides the most complex mix of micronutrients along with high quality protein. TopNutri contains iron as ferric sodium ethylenediaminetetraacetate (NaFeEDTA) [103], which shows for food vehicles high in phytate a relative bioavailability of >100% (two to three times better absorbed) as compared to ferrous sulfate, with concurrent improvement of the iron, zinc and possibly copper absorption from foods [63], [104], [105]. Iron absorption from NaFeEDTA containing food products –unlike zinc, is reported not to be influenced by higher calcium levels [106]. The authors of the study assume the indicated 7.7mg iron/7.5g recommended dose TopNutri per day [80] to relate to the compound NaFeEDTA (no information available by the company), thus in the current trial the applied dose of 3.68g TopNutri/200g portion may provide 3.8mg NaFeEDTA (≈0.5mg elemental iron [107]) The company GC Rieber Compact reviews TopNutri to have positive effects on stunting and underweight rates, to increase discharge rates of formerly severely malnourished children as compared to milk-based treatment, as well as to decrease anemia rates [108] from 63.8% to 17.6% in children aged 6 to 35 months with an increase in Hb of 1.3g/dl after four months of daily supplementation in a trial in Myanmar [109]. A small increase in iron status (reduction of soluble transferrin receptor), but no increase in Hb concentrations and similarly no decreased rates of anemia could be observed in a trial applying 2.5mg NaFeEDTA (≈0.33mg elemental iron [107]) to women and children aged 6 to 59 months in a Kenyan refugee camp over a period of 13 months [110]. Similiarly, a South African trial on school children aimed to investigate the efficacy of a low-iron MNP containing 2.5mg NaFeEDTA, applied 5d/week for 23 weeks to a high-phytate maize porridge in the intervention group. Compared with the control, the MNP was effectively increasing serum ferritin, body iron stores, WHZ, and decreased transferrin receptor, also the prevalenc of iron deficiency fell significantly. However Hb concentrations and rates of anemia were not changed during the intervention [111]. Apart from the latter low-dose trials the effectiveness of MNP in increasing Hb is well investigated. Ying Yang Bao (2.5mg iron-EDTA, 5mg ferrous fumarate; ≈2mg elemental iron [28], [107])– a MNP similar to TopNutri, has been shown in a non-controlled trial to increase Hb of Chinese children aged 6 to 23 months by 0.9g/dl over a total period of 18 months [112]. A Cochrane database systematic review [113] including eight controlled trials (Ghana, Cambodia, India, Kyrgyz Republic, Haiti, Pakistan, Kenya) lasting two to twelve months, concluded home fortification with MNP (containing 12.5mg of iron as ferrous fumarate ≈ 4.2mg elemental iron [28], 5mg zinc, and 300µg of vitamin A) to be effective in improving haematological indices, and stated MNP to be possibly comparable to daily iron supplementation (as drops or syrups). All included trials applied at least 12.5mg ferrous fumarate on a daily basis. The review also relates to studies providing intermittend or flexible MNP allocation with a lower overall daily dose (e.g. 30mg ferrous fumarate per week [114]) which have been found to be as effective on haematological response. Hereby, Hyder et al. supplemented anemic Bangladeshi children aged 12 to 24 months with either 12.5mg iron daily versa 30mg weekly (≈1.4mg/d elemental iron [28]) over 8 weeks, and reported no significant differences among the groups in the Hb increases occurred (16.1g/l versa 12.3g/l), respectively [114]. Reviewed studies significantly reduced anaemia on average by 31%, and iron deficiency by 51% as compared to no intervention or placebo in children aged 6 to 23 months. Thereby the decrease in anemia rates was equally effective in populations with different anemia prevalence, in all children aged 6 to 23 months, and independent of duration of intervention. In regard to Hb concentrations, six trials had a 5.87g/l higher mean Hb concentration at follow-up, as compared to studies with no treatment or placebo however with no obvious differences among subgroups. Two trials, that assessed effects on growth (HAZ, WAZ, WHZ), could not detect significant effects after six to twelve months of intervention, respectively. Effects on morbidity could not sufficiently be assessed, however in one trial there was a tendency of children supplemented with MNP to be more susceptiple to diarrhea or recurrent respiratory infections during the first month of intervention, whilst another trial found upper respiratory infections to be similar in MNP group (7.6%) versa placebo group (6.5%) [113]. Similarly a review including 13 studies on pre-school and school-age children found MNP to significantly reduce anemia and iron deficiency but concluded that effects on morbidity and mortality remain scarce, with no significant effects on diarrhea prevalence [115]. Hirve et al. stated sprinkles containing 12.5mg ferrous fumarate (≈4.2mg elemental iron [28]) to be similarly efficacious as compared to higher iron doses in sprinkles or drops in improving Hb in moderately anemic Indian children aged 6 to 18 months over a period of 8 weeks (mean Hb increase 1.5g/dl) [116]. The integration of MNP sprinkles in ICDS programs similarly has been found to effectively increase Hb concentrations and decrease rates of anemia from 50% to 33% in boys and from 47.4% to 34.2% in girls [27].
Findings in this trial are in conformity with studies reporting an increase in hemoglobin by MNP supplementation, however this increase was found for IG3 to be non-significant as compared to the control group.
Altogether, IG3 was not found to significantly increase Hb concentrations when compared to the CG (Table 6, Table 7). Still, IG3 remained comparable to IG1 and all other study groups throughout the intervention period. However in order to explain the lower increases in Hb or in other words the absence of the expected added value of IG3 toward IG1; probably unfavorable ratios of nutrients in the enriched supplementary meals may have had a negative impact on overall bioavailability. The risk of antagonistic interactions is deemed to be low in fortified diets, as micronutrients become chelated to dietary ligands during food digestion, implying an absorption by different pathways. Competitive interactions appear rather critical if amounts of micronutrient supplements are high and consumed without food [34]. Still, e.g. the calcium content may have possibly implied detrimental effects on iron absorption from total study meal in IG3 as outlined above. Interestingly, confirmed by the Bonferroni post-hoc test IG3 showed significant/marginal significant effects on WAZ and WHZ scores at 3rd assessment when compared to the CG. Moreover IG3 showed the best trends on reducing stunting prevalence (Supplementary Material 4), or concerning mean gains in HAZ-scores (Table 3, Figure 7). Moreover the LSD post-hoc test found IG3 superior to IG2 concerning better stunting z-scores at endline assessment (p=0.026) (Supplementary Material 8). This observation supports research that stresses the importance of an adequate supply of both, Type I and Type II nutrients for growth promotion, hereby TopNutri may provide the most holistic mix of isolated micronutrients [117]. Also for prevention or treatment of diarrhea or respiratory infections the higher zinc or vitamin A content provided may be conducive, still studies on the general application of industrial MNP remain controversial as recent studies even suggest an increased risk of diarrhea [118], [119] -probably attributable to iron promoting the growth of gastro-intestinal pathogens. The gut microbiome is increasingly discussed as crucial actor in the pathogenesis of intestinal imflammatory diseases negatively affecting iron availability [120]. Iron deficiency anemia was associated with microbiota dysbalance, and is discussed to impair the programming of infant´s physiologic systems with long-term host effects on metabolism and alteration of immunological response [121]. Moreover alterations in the mircobiome are reported to be implicated in childhood malnutrition in a mutually enhancing relationship [122]. The practical effects of interventions e.g. the current diversified study meals, on the gut microbiome need further investigation.
Predictors for the response size of hemoglobin increases
In this low-iron dose trial Hb concentrations at baseline were associated with age (baseline data previously published [24]), and over the intervention period children with lower Hb baseline values showed a better Hb response to the supplementary meals as compared to children with higher Hb baseline levels (Table 4, Table 5). Similarly, a study on pre-school children in Zanzibar reported Hb at baseline to be strongly positively associated with age, and found Hb increases to be inversely related to baseline Hb concentrations after a low-dose iron treatment (10mg ferrous sulfate ≈ 2mg elemental iron [28]) for one year [123]. A trial on Bangladeshi children 6 to 12 months found a weekly supplementation of 20mg elemental iron ineffective to increase Hb concentrations in predominantly mildly anemic children, implying the conclusion that mild anemia may have other causes than iron deficiency [124], or requires higher iron doses. The current low-dose trial seems to be much more effective for treating moderate anemia than mild anemia. The decreases observed for children with baseline Hb≥11.0g/dl (Supplementary Material 5) have to be interpreted in consideration of the small number of children in this subsample, still the prevention of anemia seems to require more holistic approaches.
In this trial, equally an increased food group consumption at baseline was associated with higher increases in Hb during the trial (Table 4, Table 5), indicating that caretakers who practice a more diversified diet at baseline may be more likely to maintain this behavior throughout the trial or even to adopt new ideas of healthy diets possibly inspired by study meals.
Aside low Hb concentrations, and an increased dietary diversity at baseline, also to be among the first-borns in a family was associated with higher increases in Hb over intervention time. This increase may be not attributable to baseline Hb in this trial, as no clear association between baseline Hb and birth order was detectable. In contrast, birth order ≥2 was associated with poor growth and anemia in children aged 10 to 18 months, living in urban slums of Mumbai [125].
Moreover a cash income >5000 INR was associated with higher Hb increases over time, suggesting that a higher cash flow improves the nutritional outcomes for children early in life. This is in line with published evidence that household wealth status is associated with dietary diversity and nutritional status of Bangladeshi children [126], as well as with hemoglobin concentration of Bangladeshi women due to increased iron intake from animal sources [127].
Weight gain
No significant differences in weight gains across the study groups were found. At the endline assessment (after 548 days) the mean weight increase for total IG (2.7kg) (not presented) matched the observed weight gain for the CG (2.7kg) (Supplementary Material 3), being equal to a mean growth velocity of 0.52g/kg/d and 0.55g/kg/d, respectively. During nutrition rehabilitation a daily weight gain is suggested with 5g/kg/d to validate the treatment in children suffering from moderate acute malnutrition [128]. Jilcott et al. conducted a five weeks feeding trial with locally produced ready-to-use food (RUF) on moderately malnourished Ugandan children aged 6-59 months and reported a mean growth velocity of 2.5g/kg/d. The peanut-soybean porridge enriched with 13.5g MLP, provided 682kcal/d, if consumed as intended, however caretakers practiced dilution [129]. Moderately malnourished Malawian children aged 42 to 60 months gained on average 2.7g/kg/d, when being fed a maize/soy flour supplementation (500kcal/d) over 12 weeks [130]. Andrew achieved with a daily supplementation of a maize porridge either enriched with 25g MLP (IG) or served plain (CG) over three months, a weight gain of 2.96 g/kg/d versa 0.55g/kg/d in SAM children aged 6 to 24 months, respectively [88]. Purwestri et al. reported weight gains of mildly wasted Indonesian children (aged 6-59 months) in a ready-to-use food (RUF) rehabilitation program for 6 weeks with 3.1g/kg/bw (daily distribution of RUF-Nias biscuits with supervision of consumption for one-third of the daily portion) and 2.0g/kg/bw (weekly distribution of full ration of RUF-Nias biscuits and weekly compliance check) [131]. Scherbaum et al. found weight gains of 1.01g/kg/bw (peanut/milk-based spread), 1.76g/kg/bw (cereal/nut/legume-based biscuits (CNL-B)), 2.31g/kg/bw (CNL-B and intensive nutrition eduction) among moderately to mildy wasted Indonesian children in a daily feeding program for an average of treatment days of 25, 33, 30 days, respectively [132].
Therewith, the observed weight gain in our study is rather low, however is in line with other trials not achieving the proposed daily weight gain of 5g/kg/bw. Moreover the lower feeding frequency and long trial duration in the current study have to be taken into account.
Higher weight gains during the first year of life have been reported to have negative effects on iron status indices [133], [134] or to be not conducive to significant changes in Hb showing even negative correlation [98]. In contrast, findings of the current study suggest a positive association of weight gains and Hb changes (controlled for age and baseline Hb concentrations) at endline assessment (r=0.146, p=0.020).
Relationship between linear growth and wasting, as well as interrelations of nutritional status, morbidity, and seasonal change
At baseline assessment of this study, increasing age in the child was reflected in decreasing HAZ- and WAZ-scores (increasing prevalence), but increasing mean WHZ-scores [24]. This positive association of age with stunting, or negative association with wasting is also reflected in the worldwide timing of growth faltering [135].
Data by Helen Keller International on children 0-59 months showed acute malnutrition to highly vary by season in Bangladesh, with the highest peak occurring in August when rice storage is getting scarce [136]. Similarly Brown et al. reported greatest nutritional deficits during monsoon season persisting until subsequent harvest. Hereby the anthropometric indicator used to define malnutrition and detect seasonal change was important to be distinguished; i.e. in the discussed study on Bangladeshi children the highest prevalence of stunting occurred several months after the periods of greatest malnutrition identified by other indicators. Food availability and rate of infectious diseases are discussed as major determinants of children´s growth in less developed countries [137]. The multi-factorial etiology of the relationship between the acute and chronic form of undernutrition (wasting and stunting) is poorly understood. Repeated episodes of wasting are discussed to imply stunting, as periods of lowest weight acquisition are followed by periods of lowest linear growth. Linear growth is reported to merely occur when the body has a minimum of energy reserves, in other words when the weight-for-height is high. Wasting reflects a condition of depleted fat and muscle mass. Leptin –produced by fat tissue, stimulates bone density, catch-up growth and the immune system. Aside a lack of micronutrients –that are required for skeletal growth rather than growth of lean tissue [138], the reason how wasting leads to stunting may be partly explained by low fat stores, however the relation remains inconclusive [139], [140], [141]. Wasting was found predictive for stunting in a study on Gambian children, confirming stunting to be an adaptation process to undernutrition [142]. Birth weight and household wealth index are reported to be negatively related to both stunting and wasting. Other common predictors for stunting were low maternal height, low maternal BMI, low frequency of antenatal care visits, higher birth order, low maternal educational level, children of mothers without decision making power regarding food, lack of minimum meal frequency, no feeding of eggs, dairy products, fruits and/or vegetables, or the delayed introduction of complementary foods, increasing age of child, and households without access to improved sanitation [139], [143], [144], [145], [146]. Acute respiratory infection was predictive for underweight, whereas diarrhea was a major driver for stunting among Indian children below five years [147]. Hence, multi-sectoral approaches have to be taken to avert underlying causes of stunting (child feeding, women´s nutrition, household sanitation), but also the distal and inter-generational drivers (adolescent marriage, women´s poor decision making power, low educational level, social exclusion and household poverty). South-Asia bears the highest global burden of child stunting (40%) [148]. Scaling-up the year-round food security by enhanced homestead food production programs already showed impact with reductions in stunting of 18% in Bangladesh [149]. Dewey reviews options for improved women´s and children´s diets and their positive impact on child growth. Still the linear growth response to improved nutrition remains heterogenous, highlighting the multi-factoral mechanisms of prenatal and post-natal growth restriction [150], also explaining why nutrition interventions alone are not necessarily implying a positive impact on growth.
Moreover rapid and simulataneous decrease in stunting and wasting rates may be challenging to achieve as any decline in stunting and underweight prevalence is commonly accompanied by a temporary increase or stagnancy in wasting [151].
Consistent with the above outlined considerations, in this study (Figure 7, Figure 8, Figure 9) underweight and wasting scores were found highest during baseline (t0), and 3rd assessment (t12) in Feb2015/16 (laggards measured in months Mar, Apr); and lowest scores were assessed at 2nd (t6), and endline assessment (t18) in Aug 2015/16 (laggards measured in months Sep, Oct), when rice storages are getting scarce close to the next harvest. Moreover during rainy season lasting from June to September, children may be more prone to infectious diseases [152], which is associated with additional nutrient requirements for the immune response. Infant feeding practices in South Asia have been shown to be highly inadequate during illness which contributes to a further deterioration of the nutritional status [153].
The prevalence of sickness at baseline was rated as high in this study as opposed to data of the NFHS-4 [24], with a perceived increase during winter season over the intervention period (Figure 10). Prevalences of sickness were invariably lower at 3rd assessment versa baseline, indicating a combined positive impact of intervention and increasing age over time on morbidity reduction in particular for IG3.
Correlation of attendance and hemoglobin
Although merely very weak correlations were detected between attendance rates throughout the study period and hemoglobin concentrations at endline assessment, the association of the effect of the attendance on the hemoglobin status of children was negative, suggesting an issue of reverse causality. Children deemed as more vulnerable by the caretakers were probably taken more often to the supplementary feeding program. The vulnerability may have been characterized by common signs of iron deficiency anemia like fatigue, pallor, or lassitude. This assumption is supported when allocating children according to their anemia status at baseline in two groups (no/mild versa moderate anemia) and considering mean rates of attendance during the 78 weeks. Children suffering from moderate anemia had a higher rate of program attendance than children with no/mild anemia (82.1% versa 75.2%), however this finding was not significant. Similarly children suffering from severe anemia were more likely to attend a feeding program in Burkina Faso, than children without severe anemia [154], altogether indicating that being more vulnerable is associated with higher attendance, and not vice versa. Reverse causality has also been suggested in other studies on breastfeeding and nutritional status, hereby mothers tended to breastfeed longer if their child was small and/or seemed to be ill [155], [156]. Besides frequent child´s illness may have implied increased program attendance. The morbidity questionnaire revealed the number of days of cold/cough being associated with the attendance rates until 3rd (r=0.289**, p<0.001) and 4th assessment (r=0.292**, p<0.001).
Correlation of baseline hemoglobin concentrations or portion size with hemoglobin
The overall consumption rate over the three IPs showed a very weak positive correlation with the Hb at the 4th assessment after adjusting for attendance rates. Moreover a very weak association was detected between the Hb changes occurred during 1st to 3rd assessment and portion sizes consumed, adjusted for attendance. Both findings indicate the intervention having a positive effect on Hb. The Hb response (during 1st/3rd and 1st/4th assessment) proofed to be negatively associated (r= -0.459**, r= -0.539, p<0.001) with baseline Hb, respectively. Similarly, oral medicinal iron supplementation and lower baseline Hb concentrations were predictors of a greater Hb response in a systematic review of 55 controlled trials with iron supplementation interventions in children [157], or the number of micronutrient sachets consumed per week was associated with increases in Hb in a home-based fortification program of complementary foods [112].
Limitations
A general limitation to the study is the unknown exact amount of nutrient losses occurred on the way from market/storage, during cooking and finally to the plate, still a careful chain of action was adhered to. Lack of Hb response in individuals may be a consequence of multiple micronutrient deficiencies, and not limited to iron supply [158]. Variant types of anemia with different etiologies like thalassemia, sickle-cell anemia, could be a major confounding factor for the current study, which were not assessed in this research. In particular in countries where the pevalence of anemia exceeds 40%, the previously assumed anemia proportion of 50% amenable to iron deficiency [159] (42% of anemia in children and 50% of anemia in women [5]) might rank much lower e.g. suggested with 14% for pre-school children, and 16% for non-pregnant women in reproductive age [159]. A holistic clinical assessment (e.g. ferritin, transferrin saturation) would do better than Hb estimation alone, still HemoCue201+ remains the suitable method to the field to diagnose anemia. Moreover the re-examination of a race specific cut-off for mild anemia is discussed due to potential genetic determinants related to the heritability of Hb, partly explaining an unchanging high burden of anemia among Indian women [160]. Several studies assessed the distribution of Hb to be lower in black people [16]. Similarly anemia during infancy is suggested as common, thus lower cutoffs may be more representative to diagnose anemia in children aged 8 months [161].
Conclusion and recommendations
After adjusting for age, baseline Hb concentrations, time between assessment points, and gender; study findings suggest the application of diversified traditional diets (IG1) to be superior toward the enrichments of 3g leaf powder/100g meal in the ratio ALP 2:1 MLP (IG2) or a for the iron content of IG2 adjusted amount of industrial MNP (1.84g TopNutri/100g meal) (IG3), in significantly increasing Hb concentrations as compared to the CG analyzed by GLM LSD post-hoc test at 2nd, 3rd, and endline assessment (confirmed by Bonferroni post-hoc at 3rd assessment) (Table 6, Table 7). Still, at 3rd and endline assessment all intervention groups showed a tendency of higher increases in Hb as opposed to the CG, with IG2 and IG3 being comparable to IG1 but also to the CG indicating a positive impact of all interventions on Hb concentrations (Figure 6). The diminished effect on Hb observed in IG2 and IG3 may be attributable to undesired nutrient interactions decreasing the bioavailability of the additionally enriched diets.
Adjusted for age, baseline z-scores, time between assessment points and gender; significant effects related to growth indices are less consistent (Supplementary Material 8, Supplementary Material 9, Supplementary Material 10). IG3 seemed to be most succeeding in improving growth indices (WAZ, WHZ) as opposed to the CG (confirmed by the conservative Bonferroni post-hoc test at 3rd checkup). Moreover IG3 showed as only intervention group tendencies of slight improvements in HAZ scores after one year of intervention (however without statistical relevance); as well as most efficiently reduced morbidity prevalence (in particular respiratory infection, but also fever and diarrhea rates).
Observed mean changes in Hb (Figure 4) have to be interpreted in relation to the low iron dose applied in this trial. Thus, the rather low increases in Hb over a long period of time are still deemed to be beneficial by the authors of this study, in particular when striving for sustainable solutions to combat anemia. The serving of a minimum of three diversified meals per week may be likely to be adopted by most caretakers on household level after nutrition counseling and interactive cooking trainings. Thus, diversified meals are a local possibility to decrease rates of anemia. Moreover to modify caregiver´s feeding practices and strengthen their resources for healthy meal preparation e.g. by maintaining kitchen garden programs which may be more cost-efficient as compared to hosting a long-term community feeding trial requiring extensive monitoring and being less appealing to caretaker´s own responsibility to make behavioral modifications. Still, for treatment of anemia therapeutic iron doses are recommended in the first line, as a rapid improvement in Hb concentrations is of priority for proper physical and mental development of the child, but still bears own limitations as outlined by the Indian National Iron Plus Initiative [162]. Moreover the enrichment with an adjusted amount of the MNP TopNutri (IG3) showed beneficial effects concerning growth indices, thus MNP may have to be considered as additional treatment approach for undernutrition. The absent effect on Hb and the inconsistency of IG3-effects on growth across different study assessment points has to be interpreted in relation to the applied dose (which was lower than recommended by GC Rieber Compact). Moreover some effects on e.g. HAZ scores which reflect the status of chronic undernutrition; need long-term interventions for improvement, thus the absence of significant effects on HAZ during the first year of study is not decisive. At endline for IG3 the beneficial impact of a holistic nutrient composition on improving stunting scores seems to slightly emerge, however without statistical relevance as opposed to the CG.
The Hb response was inversely related to baseline anemia status (Table 4, Table 5, Supplementary Material 5), suggesting low-dose trials to be most effective for treating moderate forms of anemia, with beneficial effects on mildly anemic children. For interpretation of the preventive effects of this trial on anemia the sample size was limited.
In order to alleviate micronutrient deficiencies a holistic food-based strategy –as suggested by Thompson and Amoroso [15], will embrace agricultural incentives to promote increased availability and consumption of diets composed of a variety of foods, nutrition education, implications for public health and disease control, as well as further enrichments through supplementation.
To conclude diversified supplementary meals of this trial (IG1) (dietary diversification) proofed to be a successful food-based approach to increase Hb concentrations in particular in moderately anemic Adivasi children. The enrichment of diversified diets with MNP (IG3) showed best effects on growth indices and reduction of morbidity prevalence amongst all study groups. Yet, more research is needed to particularly investigate holistic approaches for sustainable treatment and prevention of anemia and undernutrition with locally available resources.