Malnutrition is an important underlying cause of infectious disease morbidity and is related to more than half of the 10.8 million annual deaths of children younger than 5 years of age in the world (1,2). In the several last decades, millions of deaths from diarrhea have been prevented with oral rehydration solutions (3); however, diarrhea remains one of the main causes of mortality in developing countries, and it is estimated that 1.8 million children younger than 5 years of age die of diarrhea each year (4). Despite advances in knowledge about the pathogenesis and management of diarrhea, there has been little progress in the duration of episodes or incidence of diarrhea in small children living in developing countries (5). Repeated episodes of diarrhea in small children may have long-term effects on linear growth (6) and cognitive function (7), and it is a major determinant of stunting in children (8).
Infants living in developing countries have inadequate complementary foods and are most vulnerable to experience growth retardation and micronutrient deficiencies (9). Stunting and anemia are common nutritional problems in Peruvian children (10,11). Micronutrient deficiencies, such as zinc deficiency, affect immune response, increasing the risk of diarrheal infections (12).
Some proteins in the bovine milkfat globule membrane (MFGM) have been demonstrated to possess broad activities against pathogens, and a bovine whey protein concentrate enriched in the MFGM fraction may therefore help to prevent diarrhea of bacterial and viral origins (13,14). This protein concentrate contains several bioactive components including mucin (MUC1), lactadherin, folate-binding protein, lactoferrin, Immunoglobulin (Ig) G, sialic acid, sphingomyelin, and gangliosides (14). A bovine milk fraction containing MUC1 has been shown to inhibit hemagglutination of Vibrio cholerae and Escherichia coli(15). In addition, purified mucin, an MFGM constituent, was demonstrated to decrease the adherence of Yersinia enterocolitica to intestinal membranes (16). Human milk mucin components were able to bind to various rotavirus strains and prevent replication, and the ability was correlated to lactadherin (17). Furthermore, the content of lactadherin in breast milk was shown to be negatively correlated with symptomatic rotavirus infection in Mexican infants (18). The MFGM fraction also has been found to inhibit rotavirus in vitro (19). Sphingolipids, particularly gangliosides, have been shown to inhibit enterotoxins both in vitro and in vivo (20). Infant food with added sphingolipids (gangliosides) has been shown to reduce E coli counts in the stool and to increase beneficial bifidobacteria (21). Proteins that aid in the absorption of nutrients include folate-binding protein, which has been shown to facilitate the uptake of folate by intestinal cells (22) and lactoferrin, which mediates iron uptake by enterocytes (23). Taken together, it is likely that addition of the MFGM-enriched protein fraction to infant diets would reduce diarrhea, enhance nutrient utilization, and overall improve nutrient status and growth in children. The purpose of the present study was to evaluate the efficacy of a complementary food fortified with a MFGM protein fraction in diarrhea, anemia, and micronutrient status of Peruvian children.
SUBJECTS AND METHODS
The study was a double-blind randomized controlled trial of children (n = 550), 6 to 11 months old, who consumed for 6 months, an instant complementary food fortified with 1 recommended dietary allowance (RDA) of multiple micronutrients with the protein source being either the MFGM-enriched protein fraction or skim milk powder.
At entry into the study, children were assigned randomly to receive a complementary food based on cornstarch and milk with protein provided as whey protein concentrate enriched with the MFGM-enriched protein fraction (MFGM group) or an equal amount of skim milk protein (CON, control group). LACPRODAN (MFGM) is a whey protein concentrate (Arla Foods Ingredients, Viby, Denmark). It is enriched in proteins associated with the membrane surrounding the MFG (8%) and includes several other components from bovine milk, as shown in Table 1, such as IgG (5%), lactoferrin (0.2%), phospholipids (7%), gangliosides (0.2%), and sialic acid (2%). The remainder largely consists of whey proteins. Children received daily for 6 months 200 mL of the complementary food (40 g) divided into 2 servings. The nutrient content of the daily complementary food was energy 192 kcal, protein 6 g, carbohydrate 20 g, fat 9.6 g, and calcium 240 mg. The instant complementary fortified food was prepared by Arla Foods Ingredients and provided 1 RDA of iron (10 mg) as sulfate, zinc (5 mg) as sulfate, copper (0.6 mg) as sulfate, folate (35 μg), vitamin A (375 μg) as retinylpalmitate, thiamin (400 μg), riboflavin (500 μg), vitamin B12 (0.5 μg), and ascorbic acid (35 mg) (24). The sachets with the 2 test meals looked similar and were codified; the codes were opened by the investigators at the end of the study when the dataset was completed.
The study site was Villa El Salvador, a periurban community in the south part of Lima, Peru, with a population of approximately 400,000 inhabitants.
Subject Inclusion Criteria
The inclusion criteria were healthy infants, ages 6 to 11 months, born at term with a birth weight ≥2500 g, primarily breast-fed, living in the study area.
The exclusion criteria were chronic disease, serious illness or congenital malformations, severe malnutrition (National Center for Health Statistics weight/height ≤2 z scores) (25), and proven or probable milk allergy.
Eligible children were identified by home survey of the community or at the local hospital outpatient clinic. Parents were visited by field workers and received an invitation to visit the clinic for an interview with the pediatrician to explain the objectives and procedures of the study. Parents who accepted and signed the informed consent form received an appointment for the baseline clinical and nutritional evaluation of their children. After baseline evaluation, infants were randomly assigned to 1 of the 2 groups, using a computer-generated random list.
Mothers were visited daily by a field worker and received a sachet of the complementary meal (40 g); the meal was prepared with warm boiled water in a special cup, provided by the project, which had marks on it for the dilution of the product (up to 200 mL). Mothers prepared half of the meal in the mid-morning and the other half in the mid-afternoon. In 50% of the randomly selected subjects the nutritionist conducted a 24-hour recall to assess adequacy of the diet. Dietary recall was evaluated in 256 infants. In the final dietary analysis, 215 infants were included, 21 infants did not finish the study, and 20 infants were excluded for the following reasons: received their meal at a day care center, were sick during the interview, did not have a regular feeding pattern (eg, birthdays, other celebrations), their mothers did not recall well, or they had extreme values. The food household measures were transformed into grams and codified according to the Instituto de Investigación Nutricional (IIN) Peruvian food composition tables (unpublished) to estimate the intake of energy and nutrients for 24 hours. A majority of the children were still being breast-fed at the start of the study. To estimate the adequacy of the diet we considered recommendations on complementary feeding of young children in developing countries (9), and for micronutrients we considered Food and Agriculture Organization/World Health Organization recommendations on human vitamins and mineral requirements (26). Dietary intake and nutritional status change considerably during infancy; therefore, we analyzed the entire group and the 6- to 8- and 9- to 11-month-old groups separately.
Mothers were interviewed twice per week in their homes by field workers who recorded information on diarrhea morbidity, consumption of the product, and feeding patterns, following a methodology from previous studies in Peru (27,28).
At baseline and at the end of the study, the infants were evaluated by the study team. The evaluation included clinical assessment by a pediatrician; anthropometric evaluation: weight, length, mid-upper arm circumference, head circumference, and tricipital, bicipital, and subscapular skinfolds; dietary assessment; and a venous blood sample (4 mL) for assessment of hemoglobin, serum ferritin, zinc, and folate. Every month, and more often if necessary, children were examined by a pediatrician, and a nutritionist measured weight and length every month. Data were specially codified to ensure confidentiality, and investigators and personnel at the clinic were not aware of the study groups.
Definitions of Outcomes
Anemia was defined as hemoglobin <110 g/L according to the International Nutritional Anemia Consultative Group (29) and <105 g/L as suggested for this age group previously (30). Iron deficiency was defined as serum ferritin <10 μg/L, zinc deficiency as serum zinc concentration <10.7 μmol/L, and folate deficiency as serum folate <6.8 ng/mL (9,12,30,31). Biochemical analyses were conducted only for children who had the 2 blood samples. For folate, 480 children were included in the final dataset, for ferritin 445 children, and for zinc analyses 494 children.
Diarrhea was defined as >3 loose stools or 1 bloody stool within the last 24 hours and severe diarrhea as ≥6 liquid or loose stool in the last 24 hours plus 1 of the following signs: documented fever or dehydration, demanded health care, or found blood in stool. Persistent diarrhea was defined as a diarrheal episode lasting >14 days. Longitudinal prevalence was defined as the percentage of days that a child has acute diarrhea out of the days measured. Incidence was reported as the number of episodes of diarrhea divided by the number of observation days per child per year. New episodes were separated by at least 2 diarrhea-free days.
Hemoglobin was measured directly at the outpatient clinic with a Hemocue instrument (Hemocue, Ängelholm, Sweden); serum was separated and kept frozen for later analysis. Serum zinc was measured following wet ashing with concentrated nitric acid (32) using a SOLAAR M series atomic absorbance spectrometer (Thermo Electron Corp, Waltham, MA). Serum ferritin was measured by radioimmunoassay (Diagnostic Products, San Diego, CA). Folate in plasma was quantified by radioimmunoassay using a radioassay kit (MP Biomedicals; Solon, OH). Biochemical analyses were done at the Department of Nutrition, University of California, Davis.
Stool samples were taken during the diarrheal episodes; samples were placed in stool culture transport vials (Cary-Blair) and sent to the IIN for microbiological analyses. For isolating Salmonella, Shigella, E coli, and V cholerae, the samples were cultured directly onto commonly used selective enteric media (SS agar, XLD agar, Mac Conkey agar, and TCBS agar plates) enriched in selenite broth and alkaline peptone water. For isolating Campylobacter, samples were cultured directly onto Butzler agar. E coli isolates were analyzed in the Laboratory of Enteric Diseases and Nutrition at University Cayetano Heredia, Lima, Peru, using a Multiplex Real Time SYBR Green–based polymerase chain reaction for detection of enterotoxigenic (ETEC), enteropathogenic (EPEC), Shiga toxin–producing (STEC), enteroinvasive (EIEC), enteroaggregative (EAEC), and diffusely adherent E coli (DAEC) (33). The presence of rotavirus was assessed at the IIN by an enzyme immunoassay for the detection of rotavirus in human fecal specimens from IDEIA Rotavirus (Dako Cytomation, Carpinteria, CA).
For parasite analysis, merthiolate-iodine-formaldehyde was used for preserving, staining, and fixing the stool specimens (34). Enzyme immunoassay was used for detection of Giardia-specific antigen (GSA 65) in aqueous extracts of fecal specimens (ProSpecT Giardia Microplate Assay, Alexon-Trend, Lenexa, KS).
Data were analyzed using SPSS version 12.01 for Windows, and STATA (version 11.0, STATACorp, College Station, TX) (35). The primary goal of the analysis was to identify differences between groups with regard to diarrhea morbidity. Analysis was based on intention to treat. Initially, the data were analyzed as a 2-factor analysis with interaction, and when the interaction was found to be significant (P < 0.05), an analysis of the subgroups was performed. Age and sex interactions also were analyzed.
Chi-square tests were used to compare categorical variables such as socioeconomic variables and nutritional deficiencies, and analysis of variance tests were used to compare continuous variables between groups; P values <0.05 were considered significant. To compare mean duration of diarrhea episodes, we used analysis of variance. To compare prevalence rates we used Poisson distribution tests. To analyze odds ratio (OR) for diarrhea we used a generalized estimating equation model (36).
Multivariate logistic regression models were also run to estimate risk of anemia at the end of the study. To compare dietary intake between groups we did analysis of covariance controlling for initial dietary intake and age.
The protocol was approved by the ethics committees of IIN, and the University of California, Davis, and it was authorized by the Instituto Nacional de Salud from the Ministry of Health, Lima, Peru. Parents of participant children signed the informed consent form at entry.
General Socioeconomic Status and Demographic Characteristics
A total of 550 infants were enrolled and 499 (91%) of them completed the study that was conducted during 2004–2005. The reasons for dropping out of the study are shown in Figure 1; they were similar in the 2 groups. As shown in Table 2, the MFGM and control groups also were similar and homogenous with regard to variables of general socioeconomic and demographic status (P > 0.05), except for proportion of infants with hemoglobin <105 g/L and potable water facilities; therefore, analyses were controlled for these variables.
Nutrient Intake Before and After Addition of Complementary Food
All of the infants (100%) were primarily breast-fed, but only 27% of them were exclusively breast-fed (as defined by the World Health Organization) until 6 months of age, whereas 15% were given some infusions/juice, 23% other milk, and 35% other food; there were no significant differences between the groups. Mean age for start of complementary foods was 5.4 months in both groups. The regular diet consumed by the infants at entry into the study was inadequate in energy, calcium, iron, zinc, and retinol as compared with present recommendations (9), but there was no difference between groups (Table 3).
Protein and folate intake met recommendations at entry and at the end of the study. At the end of the study, energy and micronutrient intake had increased in both groups and was significantly higher than at baseline for energy, iron, zinc, calcium, and retinol; however, their regular diet did not cover present recommendations for these nutrients. The final dietary intake of iron and retinol was higher in the MFGM group than in controls (P < 0.05). Consumption of the test diet was satisfactory during the entire study period; mean daily intake in the MFGM group was 28.7 ± 10.4 g and 29.1 ± 10.6 g in the control group (not significant), which corresponded to 72% and 73%, respectively, of the 40 g offered each day. The test diet was the main source of micronutrients. With consumption of the regular diet plus the complementary meal, the present recommendations for energy and micronutrients were met by both groups.
Hematological and Biochemical Indicators
There were no significant differences between groups for initial mean hemoglobin values in the 6- to 8-month-old groups (104.6 and 104.8 g/L in the MFGM and control groups, respectively), but there was a significant difference between groups in the 9- to 11-month-old group (101.7 and 104.4 g/L, respectively, P < 0.032) (Table 4). Within groups, final hemoglobin values were higher than initial values (P < 0.01). Comparing by treatment groups, final hemoglobin values did not differ in the 6- to 8-month-old group, 108.2 and 109.1 g/L in the MFGM and control groups, respectively, but they did differ in the 9- to 11-month-old group, 108.0 in the MFGM and 111.1 g/L in the control groups (P < 0.01). Analysis of covariance with a block design with initial hemoglobin as covariate and controlling for age showed a difference in final hemoglobin between groups with higher mean values in the control group (109.9 vs 108.1 g/L in the MFGM group (P = 0.049). Initial prevalence of anemia (using 105 g/L as cutoff) was 57% and 47% in the MFGM and control groups, respectively (in the 6- to 8-month-old cohort 52% and 46%, and in the 9- to 11-month-old cohort, 65% and 51%), but had decreased significantly at the end of the study to 28% and 23% in the MFGM and control groups, respectively, but there was no difference between groups; final prevalence of anemia in the 6- to 8-month-old group was 27% and 25% in the MFGM and control groups, respectively, and 30% and 21% in the 9- to 11-month-old group. Controlling for initial anemia and age, the Cochrane test showed no significant difference between groups in anemia. A multivariate logistic regression model for final anemia showed a 69% protection from anemia in all of the children who were not anemic at entry into the study, OR 0.31 (0.16–0.58) (P < 0.001), and an increase in anemia risk in all of the children who had have episodes of diarrhea, OR 1.18 (1.06–1.32) (P = 0.02).
Serum ferritin values were not normally distributed and were therefore log transformed. There were no differences between groups at entry or at the end of the study (Table 4). At entry into the study, iron deficiency (defined as serum ferritin <10 μg/L) was 32% and 38% in the MFGM and control groups, respectively. The prevalence of iron deficiency did not change much; at the end of the study, 24% and 25% in the MFGM and control groups, respectively, were iron deficient and there were no differences between groups.
There were no significant differences between groups in serum zinc at entry or at the end of the study for either the 6- to 8-month-old group or the 9- to 11-month-old group (Table 4). Serum zinc values were not normally distributed and were therefore log transformed. We used the t test for comparisons because the variance was homogenous. There was a high prevalence of zinc deficiency among the infants at entry into the study using the 10.7 μmol/L cutoff suggested by the International Zinc Nutrition Consultative Group (12), 82% and 78% in the MFGM and control groups, respectively. At the end of the study, this prevalence was similar, 80% and 83%, in the MFGM and control groups, respectively.
There were no significant differences in serum folate between groups at entry into the study or at the end of the study; however, mean values were lower at the end of the study in both groups and had decreased from 15.3 to 12.9 in the MFGM group and from 16.3 to 13.4 μg/L in the control group (Table 4). The prevalence of folate deficiency at entry (serum folate <6.8 μg/L) was 5.3% and 4.7% in the MFGM and control groups, respectively (Table 4). At the end of the study, this prevalence had increased slightly to 10.6% in both groups, but it was not significantly different between groups.
During the study there were a total of 1549 diarrhea episodes, 750 in the MFGM group and 799 in the control group; a total of 45 infants (9%) did not have a diarrhea episode during the study period (21 in the MFGM group and 24 in the control group). Longitudinal prevalence of diarrhea during the study was 3.84% in the MFGM and 4.37% in the control group (P < 0.05). The mean (± standard deviation [SD]) duration of episodes of diarrhea was 2.38 (1.5) in the MFGM and 2.36 (1.4) days in the control group, and there was no difference between groups. Of the total number of episodes, 1478 (95%) had a duration of ≤7 days and 17 (1.1%) lasted >14 days (persistent diarrhea). The highest number of diarrhea episodes was observed during the summer months (December–April). There was no significant effect of exclusive breast-feeding as compared to partial breast-feeding on diarrhea outcomes. There were no differences between groups in weight/height, height/age, or weight/age nor did these measures influence diarrhea outcomes.
The incidence of all of the episodes of diarrhea per child per year was 5.81 in the MFGM group and 6.38 in the control group; the unadjusted and adjusted ORs were not significant (Table 5). Incidence of severe diarrhea was 1.63 (MFGM) and 1.84 (control) episodes per child per year, with no significant difference between groups. In longitudinal multivariate regression modeling for diarrhea outcomes, adjusting for initial anemia (hemoglobin < 105 g/L) and potable water facilities, there was a 46% reduction in episodes of bloody diarrhea in the MFGM group as compared to the control group, with an OR of 0.54 (0.31–0. 93) (P = 0.025) (Table 5).
The most common pathogen isolated during the diarrhea episodes was diarrheogenic E coli (45% of pathogens), and the most common serotypes were EAEC (14% and 13% in MFGM and control groups), EPEC (10% and 11%, respectively), and DAEC (5% and 12%, respectively) (not significant between groups). Campylobacter was diagnosed in approximately 25% of the samples and with no difference between treatment groups. Both E coli and Campylobacter suggest food/water contamination. The frequency of rotavirus diarrhea in the MFGM group and the control group was 8.0% and 9.5%, which was not significantly different between groups. Most cases of rotavirus diarrhea occurred during the winter. Giardia lamblia and Blastocystis hominis were the most common parasites (8% and 11%, respectively). There were no significant differences between groups. In 40% of the samples analyzed, no pathogens were identified.
The present study showed that adding whey protein concentrate enriched with the MFGM fraction to complementary food significantly reduced the likelihood of infants having bloody diarrhea. It also reduced the prevalence of acute diarrhea, but there was no effect on the duration of diarrhea. Bloody diarrhea has been associated with Shigella(37) and some serotypes of diarrhoegenic E coli(38). The keystone for the management of bloody diarrhea is the early use of effective antimicrobial treatment; however, increased antimicrobial resistance of diarrheogenic E coli has been reported in Peru, as in many developing countries (39,40). Therefore, dietary supplements to prevent diarrhea are likely to be more cost-efficient and sustainable than treatment with antibiotics.
Diarrhea is a global public health problem, and although several approaches have been evaluated, to date only zinc supplementation has consistently shown a significant reduction in diarrhea incidence and duration (41). The effect of bioactive milk components (MFGM) on diarrhea that we observed could be attributed to a direct effect on gut microflora, to enhanced immune function, or by components functioning as a decoy for pathogens. Several studies have shown inhibitory effects of individual bioactive proteins, such as MUC1, lactadherin, IgG, and lactoferrin, on pathogens (13–19). Lactoferrin also has anti-inflammatory activity, possibly limiting the effects of infection and reducing bloody diarrhea. Other studies in different settings are needed to confirm these findings and to study underlying mechanisms.
In the study population, intakes of energy, calcium, iron, and zinc from complementary foods were considerably lower than the recommendations for infants. Inadequate intakes were reflected by a high prevalence of anemia, iron, and zinc deficiency. Although the primary purpose of the present study was to evaluate the efficacy of bioactive milk proteins in diarrhea, we had hoped to improve the micronutrient status in this population by adding 1 RDA of micronutrients to their daily diet. Because the proteins and lipids present in the MFGM fraction also have the potential to facilitate nutrient absorption, we evaluated whether the addition of MFGM would improve the nutritional status of children. Intakes of energy, iron, zinc, folate, retinol, and calcium met or exceeded the present recommendations after daily consumption of the test diets; although we observed improvements in hemoglobin and iron status, there were no improvements in zinc or folate status.
The addition of 1 RDA of zinc (5 mg/day) to the test meals was inadequate to increase serum zinc significantly and to decrease the prevalence of zinc deficiency. It is possible that some component in the subjects’ regular diet limited the absorption of zinc or that the need for zinc is higher in this population. Thus, larger amounts than 1 RDA of zinc may be required to improve zinc nutrition in this population. In a study of 6-month-old Indonesian infants given daily supplements of zinc (10 mg), plasma zinc increased after a 6-month intervention (42), but the supplements were given in a fasted state and the absence of food can be expected to increase the absorption of zinc. In Sweden, 12-month-old infants fed according to present recommendations were found to have a high prevalence of low plasma zinc (36%) using the same cutoff (43). In another study in Sweden of infants fed a cereal-based complementary food together with other foods, 18% of the infants had low plasma zinc at the start of the study (6 months old), but 27% had low values at 12 months old (44). These studies show that low zinc status is common at 12 months old, even in affluent settings, suggesting that zinc requirements in this age group may be underestimated.
The high prevalence of anemia is similar to what has been observed in national surveys (10,11). Our intervention significantly reduced anemia and improved iron status in both groups. The reasons for the high prevalence of anemia at 6 months of age are not completely known, but it may be caused by low iron endowments at birth resulting from a high prevalence of maternal iron deficiency anemia in this population (10,11), and repeated episodes of diarrhea. Early introduction of complementary foods with low iron content and low bioavailability increases the risk of anemia in infancy. During the 6-month intervention period, however, overall anemia prevalence fell from 49% to 26% in both groups, showing that the iron provided was well used. It should be noted that in the study of Indonesian infants (42), supplementation with iron in the form of drops reduced the prevalence of anemia from 41% at 6 months to 25% at 12 months, whereas 44% of the infants in the placebo group were anemic at 12 months. It is well known that if iron is not provided at this age, the prevalence of anemia increases because of a lack of iron in the diet and the presence of factors inhibiting iron absorption (45). As can be seen, diet alone in this population provided only 24% of recommendations for iron intake at the start of the study and 68% at the end, clearly inadequate to meet infants’ iron needs. Thus, providing 1 RDA of iron in the complementary food had a beneficial effect on the prevalence of anemia. At the end of the study, 26% of the children were still anemic. Similar proportions remaining anemic after intervention have been noted in several studies on iron supplementation/fortification of infants and children in other countries (46–49). Multiple regression analyses showed that the risk of being anemic after the intervention was higher in children who were anemic at entry and in children with 1 or more episodes of diarrhea; therefore, prevention of diarrhea would contribute to reduction of anemia.
The addition of 1 RDA of iron to the test diets did not alter iron status in the 6- to 8-month-old group, but iron deficiency decreased to 26% in the 9- to 11-month-old group. In the study of 12-month-old infants in Sweden, 26% were also iron deficient (44) in spite of ample intakes of iron, suggesting that iron intakes meeting recommendations are not sufficient to eliminate iron deficiency. Iron absorption is known to be inversely correlated to iron status, and it is therefore likely that the 9- to 11-month-old group, which had lower serum ferritin at entry, had a higher capacity to absorb dietary iron than the 6- to 8-month-old group. It has been shown that mechanisms regulating iron absorption are immature in infants at 6 months of age, but respond to iron intake/status at 9 months of age (45). It is not known when this maturation of iron homeostasis occurs, but the fact that infants in the 6- to 8-month-old group did not show any change in iron stores may be explained by this observation.
Serum folate decreased significantly during the study period, and the prevalence of folate deficiency in all of the children (as defined by serum folate <6.8 μg/L) increased from 5% to 11%. Folate intake from their diet alone in relation to recommended intakes decreased from the start to the end of the study; however, with the folate-fortified test diets, intakes were considerably higher than recommendations. It has been shown that folate deficiency is common in Brazilian infants and that weaning increases this proportion (47). Because our test diets were fortified with 1 RDA of folate, our results suggest that folate was poorly absorbed, that infant folate requirements have been underestimated, or that the cutoff used is not appropriate for this age.
In conclusion, our results show that addition of a whey protein concentrate enriched with MFGM to complementary food given to infants reduced the likelihood of having an episode of bloody diarrhea and the prevalence of diarrhea. Addition of 1 RDA of iron and other micronutrients to complementary food reduced the prevalence of anemia, whereas iron and zinc deficiency remained high and folate deficiency increased, suggesting that higher levels of fortification of these nutrients may be required in populations such as the one we studied.
We thank the parents and children who participated in the study, and the study team, health authorities from the district area of Villa El Salvador and the regional health area, DISA Lima Sur, Red de Salud VES-LPP, the Peruvian Ministry of Health, and the health personnel of the CMI San Jose, Villa El Salvador, Lima. We also thank Teresa Garcia and Deysi Galvez for statistical analyses and Dr Theresa Ochoa from Cayetano Heredia University, Lima.
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