Malnutrition and parasitic diseases are within the major issues in rural areas in developing countries. In this study, the nutritional status, dietary intake including mineral absorption inhibitor (phytate), hematological indicators and trace element status (zinc, iron) were evaluated and associated to the presence of intestinal parasites in a group of children from a rural area of Bolivia. The results showed that 96% of the children had intestinal parasites; 7 types of parasites ( Ascaris lumbricoides, Giardia lamblia, Ancylostoma duodenale, Entamoeba histolytica, Entamoeba coli, Trichuris trichiura, Strongyloides stercolaris) were identified. Anthropometric measurements indicated that 37% of the children were stunted and 17% were underweight. Iron and zinc intake showed that 34% and 30% of children had inadequate intake of these nutrients respectively. Phytate: zinc molar ratios were between 6.5 and 21, and from 6.2 to 15 for phytate: iron, indicating that the absorption of zinc and iron might be compromised by the level of phytate in the diet. The serum zinc was below the lower cut-off in 87% of the children, indicating zinc deficiency. Moreover, a multiple regression model showed the significant effect of the presence of the parasite Giardia lamblia and phytate intake on the serum zinc levels. Regarding the iron status, 30% of the children presented with anemia and about 66% had iron deficiency; a simple linear regression model showed the significant negative effect of the presence of the parasite Ancylostoma duodenale on iron status. In conclusion, the levels of zinc and iron, which were low in this child population, were greatly affected by the presence of intestinal parasites; in addition, the consumption of plant-based diets with high levels of phytate also impaired the zinc absorption.
Micronutrient deficiencies are a major health problem in developing countries; the conditions can be aggravated by poor diets and infectious diseases which create a complex cycle that is difficult to overcome, especially so in vulnerable populations such as those of pre-schoolchildren because of their higher growth requirements [
Zinc and iron are essential micronutrients for human growth and development, as well as for the maintenance of the immune system, participating in many enzyme and biological processes [
Zinc and iron deficiencies are also caused by the intake of food with low bioavailability of these essential trace elements, due to the presence of absorption inhibitors such as phytates, polyphenols and oxalates, mainly found in plant-based diets. Phytate is one of the main inhibitors; it has the ability to bind divalent minerals such as Zn2+ and Fe2+, preventing their absorption by the body [
In the present pilot study, the nutritional status, including anthropometric indicators, and dietary intake with focus on minerals and mineral absorption inhibitor-phytate, trace elements status (zinc and iron) and hematological indicators were investigated in a group of children from a tropical rural area of Bolivia. The presence of intestinal parasites was also evaluated. Furthermore, regression models were applied to assess the effects of the presence of parasites, and the intake of phytates, on the levels of zinc and iron in serum.
The pilot study was carried out in a rural area named Ibuelo, located in Chapare, approximately 160 km east of Cochabamba, Bolivia. Forty-six children from 4 to 13 years old were enrolled in this pilot study. With the help of the local health center we made contact with the school, and the principal of the school randomly (one child after five in the school list) assigned children of different ages. Prior to any enrollment, the parents of the children were informed about the objectives of the study and a statement of consent was signed, and permission was obtained. The exclusion criteria were: children presenting any diseases at the moment of the study, children with diarrhea, and children taking drugs or mineral-vitamin supplements. The study protocol was approved by the Ethics Committee of the Albina Patiño Pediatrics Hospital, Cochabamba Bolivia.
The children were lightly dressed and without shoes when measurements of height, with a portable stadiometer ±1 mm, and weight using a digital electronic scale 150 kg ± 0.1 kg (Omron HBF-400) were done, according to standardized procedures [
The dietary intake of the children was assessed during two consecutive days by the Food Photography 24-hour Recall method (FP24-hR) previously validated and described in detail [
The intake of energy, protein, fat, carbohydrates, fiber, calcium, iron, phosphorus, zinc, cooper, thiamin, riboflavin, niacin, folate, and vitamins A, C, E, B6, and B12 were calculated. These nutrients were selected in order to shed light on possible deficiencies or excesses. The inadequacy of energy intake was evaluated as intakes below two-thirds of the energy expenditure [
The enrolled children were provided with sterile clean leak-proof stool cups for collection. Diagnosis of intestinal parasites was made following standardized procedures of sedimentation and microscopic examination of stool [
Blood samples (5 ml) were drawn from fasting children from the antecubital vein, into trace element-free, heparin-container Vacutainer tubes (Becton Dickinson AB, Stockholm, Sweden).The procedures of blood collection and storage follow the guidelines given by IZiNCG [
Serum zinc was quantified by flame atomic absorption spectrometry (Model 2280, Perkin Elmer Corporation, Norwalk, CT, USA). Before analysis the samples were diluted 10 times with deionized water [
Hemoglobin, hematocrit and red blood cells (RBC) were determined by a microhematocrit reader (Microhematocrit system, Model StatSpin CSD2, CritSpin, Iris International Inc., USA). The mean corpuscular volume (MCV) was calculated by dividing the hematocrit by the total number of red blood cells (Hematocrit/RBC) and multiplied by 10. The mean cell hemoglobin concentration (MCHC) was calculated by dividing the hemoglobin by the hematocrit (hemoglobin/hematocrit).
White blood cells were analyzed in a Neubauer hemocytometer by microscopic examination. Neutrophils, basophils, lymphocytes, eosinophils and monocytes were counted through the morphological evaluation and identification. The count was done with 100X magnification under oil immersion (Biological microscope Model Olympus CX31, OLYMPUS Latin America INC.).
The Statistical Package for Social Sciences (SPSS) version 18.0 (SPSS Inc., IBM corporation 2010, www.spss.com) was used to perform the statistical treatments. The significance level was set up at P values < 0.05. Normality of the data was assessed by Shapiro-Wilk test, and measures of skewness and kurtosis. The majority of the variables do not follow a normal distribution (P < 0.05), thus the results are presented as median, minimum and maximum values. Spearman’s correlations were computed to study the association between serum zinc and iron with the corresponding micronutrient intakes and with phytate intake.
Simple and multiple linear regression analyses were computed to evaluate the effect of the number and type of parasites found in the children and the effect of phytate and mineral intake, on the levels of zinc and iron in serum. The total group of children was divided into children with specific parasites on the one hand, and the group without these parasites, on the other hand, which was used as a control group, where Man-Whitney U test was used to compare and evaluate differences in nutrient intake, anthropometric indicators, hematological and trace element status between the groups (with and without the parasites). Additionally, one way ANOVAs were used, after dividing the total group of children in subgroups with zero to n-number of different species of parasites found, to examine the effect of polyparasitism on the dietary intake, anthropometric indicators, hematologic and trace element status.
Forty-six children participated in the study: 20 boys and 26 girls; the age range was from 4 to 13 years old. Anthropometric measurements are shown in
The dietary evaluation showed that the energy distribution was within the recommended values from WHO [
Anthropometric index | Median | Minimum | Maximum | n (%) |
---|---|---|---|---|
Age, y | 8 | 4 | 13 | |
Height, cm | 120 | 94 | 154 | |
Weight, kg | 22.5 | 11.3 | 44.0 | |
HAZa | −1.49 | −3.11 | 0.40 | |
Stunting (HAZ ≤ 2SD) | 17 (37) | |||
Normal | 29 (63) | |||
WAZb | −1.02 | −3.05 | 0.78 | |
Wasted (WAZ ≤ 2SD) | 8 (17) | |||
Normal | 38 (83) | |||
BMIAZc | −0.21 | −2.08 | 0.93 | |
Underweight (BMIAZ ≤ 2SD) | 8 (17) | |||
Normal | 38 (83) | |||
Overweight (BMIAZ > 2SD) | 0 (0) |
aHAZ, z-scores height-for age; bWAZ, z-scores weight-for age; cBMIAZ, z-scores body mass index-for-age.
are adequate for all the children. However, fiber intake is inadequate in 100% of the children, as well as higher levels of inadequacy were found for vitamin E, A and calcium (higher than 90%), intake of folate and vitamin B12 were inadequate in 65 and 39% of the children respectively. Regarding zinc and iron intake, the diet of the children showed to be inadequate to meet the requirements in 30% and 34% of the children respectively.
Phytate intake was from 0.32 to 1.42 g/d, with corresponding high molar ratios of Phy:Zn and Phy:Fe likely to impair mineral absorption, 22% children presented Phy:Zn higher than 15 and 100% of them had Phy:Fe higher than the desirable value 1. The high level of phytate was due to some of the main foods in the diet being cereals and legumes, which are known to have high levels of phytate; tubers and roots are the main staple food and also contain phytate.
Intestinal parasites were found in 96% of the children. Seven types of parasites were identified; Giardia lamblia (n = 8, number of children presenting this parasite), Entamoeba histolytica (n = 8), Entamoeba coli (n = 5), Ancylostoma duodenale (n = 9), Ascaris lumbricoides (n = 28), Trichuris trichiura (n = 19) and Strongyloides stercolaris (n = 2). Forty-two percent of the children had one type of parasite, 37% had two types of parasites, 13% had up to three types of parasites and 4% had four types of parasites (
The levels of zinc and iron in serum are shown in
The white blood cell counts were low in 22% of the children; 63% of the children presented low levels of lymphocytes, 100 % of them presented high levels of eosinophils, and neutrophils were lower in 33% of the children; basophils were at the normal level for 98% of the children and monocytes were lower than the normal level for 100% of the children (
Nutrient | Median | Minimum | Maximum | % children below EARa |
---|---|---|---|---|
Energy, MJ/d | 4.6 | 3.3 | 6.4 | 43b |
Protein, g/d (%E)c | 41 (14) | 24 (10) | 54 (18) | 0 |
Fat, g/d (%E)c | 23 (19) | 11 (11) | 46 (27) | 26 |
Carbohydrates, g/d (%E)c | 185 (68) | 135 (60) | 258 (77) | 0 |
Fibre, g/d | 12 | 7 | 17 | 100 |
Calcium, mg/d | 311 | 202 | 558 | 96 |
Iron, mg/d | 7.9 | 5.5 | 12.5 | 34 |
Magnesium, mg/d | 172.0 | 127.8 | 253.3 | 11 |
Phosphorus, mg/d | 675 | 374 | 1094 | 40 |
Zinc, mg/d | 5.6 | 3.6 | 7.5 | 30 |
Copper, mg/d | 0.78 | 0.53 | 1.08 | 0 |
Vitamin C, mg/d | 48 | 12 | 159 | 17 |
Thiamin, mg/d | 0.80 | 0.49 | 1.59 | 4 |
Riboflavin, mg/d | 0.73 | 0.39 | 1.51 | 26 |
Niacin, ug/d | 11.1 | 7.1 | 22.3 | 9 |
Vitamin B-6, mg/d | 1.07 | 0.71 | 2.29 | 0 |
Folate, ug/d | 172 | 98 | 267 | 65 |
Vitamin B-12, ug/d | 1.25 | 0.46 | 2.57 | 39 |
Vitamin A, µgRAE/d | 176 | 47 | 341 | 91 |
Vitamin E, α-Tocmg/d | 1.9 | 1.1 | 5.6 | 100 |
Phytates, g/d | 0.59 | 0.32 | 1.42 | |
Molar ratio Phy:Zn | 11 | 6.5 | 21 | (22% >15)d |
Molar ratio Phy:Fe | 6.2 | 3.7 | 15 | (100% >1)d |
aPercentage of children with inadequate nutrient intake, assessed by the estimated average requirements (EAR) cut-point method, using EAR standard values according to sex and age from WHO [
Strong negative correlations were found between serum zinc and phytate intake (r = −0.439, P = 0.01), as well as with the molar ratio Phy:Zn (r = −0.301, P = 0.05), suggesting that the phytate level in the diet of the children impairs the zinc absorption. The multiple linear regression analysis for evaluating the significance of the effect of phytate and zinc intake on serum zinc, indicated that phytate intake had a significant negative effect (B1 = −20.0 ± 6.9 µg/dl, r = −0.458, P = 0.006) on the level of serum zinc; the model shows that serum zinc is decreased by 20.0 ± 6.9 µg/dl for every additional unit (g/d) of phytate in the diet. The effect of zinc intake on the serum Zn was not significant (P = 0.824).
The simple linear regression analysis for evaluating the effect of each specific parasite on the levels of zincandironin serum showed that the parasites: A. lumbricordes, E. hystolytica, E. coli, T. trichiura and A. stercolaris have no significant effect on the serum levels of these trace elements; neither were any significant differences found when comparing dietary intake, anthropometric indicators and trace element status between children with or without each specific of these parasites.
However, simple linear regression models for G. lamblia and A. duodenale showed a significant effect on the
Intestinal parasites | n (%) |
---|---|
Number of different parasitesa | |
No parasites | 2 (4) |
One type of parasites | 19 (42) |
Two types of parasites | 17 (37) |
Three types of parasites | 6 (13) |
Four types of parasites | 2 (4) |
Type of parasiteb | |
Protozoa | |
Giardia lamblia | 8 |
Entamoeba hystolytica | 8 |
Entamoeba coli | 5 |
Helminthes | |
Ascaris lumbricoides | 28 |
Trichuris trichiura | 19 |
Ancylostoma duodenale | 9 |
Strongyloides stercolaris | 2 |
aFrequency of children with none, 1, 2, 3 or 4 parasites; bNumber of children with the specific type of parasite.
Median | Minimum | Maximum | %below reference | %normal range | %above reference | Reference valuesa | |
---|---|---|---|---|---|---|---|
Hemoglobin, g/dl | 10.5 | 5.7 | 14.6 | 72 | 28 | - | 11.5 - 15 |
Hematocrit, % | 33.0 | 18.0 | 46.0 | 61 | 11 | 28 | 33 - 36 |
Mean corpuscular volume (MCV), fl | 93.0 | 93.0 | 94.0 | - | - | 100 | 79 - 80 |
Mean cell hemoglobin concentration (MCHC), g/dl | 32 | 32 | 32 | - | 100 | - | 32 - 36 |
Red blood cells, units × 106/mm3 | 3.53 | 1.93 | 4.92 | 63 | 26 | 11 | 3.7 - 4.8 |
White blood cells, units × 1000/mm3 | 5.3 | 2.4 | 14.4 | 22 | 63 | 15 | 4.0 - 10.0 |
Lymphocytes, % | 33.0 | 17.0 | 46.0 | 63 | 34 | - | 34 - 50 |
Eosinophils, % | 23.0 | 4.0 | 38.0 | - | - | 100 | 3 |
Neutrophils, % | 43 | 27 | 62 | 33 | 56 | 11 | 42 - 59 |
Basophils, % | 1 | 1 | 2 | - | 98 | 2 | 0 - 1 |
Monocytes, % | 1 | 1 | 3 | 100 | - | - | 4 - 5 |
Serum Zn, ug/dl | 55.0 | 40.9 | 81.4 | 87 | 13 | - | <65 |
Serum Fe, ug/dl (n = 44)b | 32.9 | 11.0 | 95.1 | 84 | 16 | - | <65 |
Serum TIBCc, ug/dl (n = 44)b | 319 | 139 | 763 | 2 | 68 | 30 | 240 - 400 |
Serum %TSd, % (n = 44)b | 9.7 | 2.2 | 28.5 | 66 | 33 | - | <16 |
aReference values for children, compiled by IZiNCG [
levels of serum zinc and iron respectively. The model indicates that the presence of G. lamblia had a significant negative effect (B = −7.4 ± 3.4 µg/dl, r = −0.310, P = 0.038) on the serum zinc of the children. Serum zinc in children with G. lamblia is 7.4 µg/dl lower than serum zinc in children without this parasite. As has previously been shown, phytate intake also had a significant effect on serum zinc; therefore, a multiple linear regression model was applied to evaluate the extent to which the phytate intake and the presence of G. lamblia effect on serum zinc levels; this model showed that the serum zinc is significantly affected by the phytate intake (B1 = −15.8 ± 4.4 µg/dl, r = −0.482, P = 0.001) and also by the presence of G. lamblia (B2 = −6.6 ± 3.1 µg/dl, r = −0.310, P = 0.035) (
Additionally, the test Man-Whitney U was used to compare the parameters of the dietary intake, anthropometric indicators and trace element status in the group of children infected with G. lamblia (n = 8) and the group of children without this parasite (n = 38). The mean serum zinc level was significantly lower (P = 0.026) in the group of children with G. lamblia (49.6 µg/dl) compared to the other group (56.9 µg/dl). There were no significant differences in the comparison of zinc intake (P = 0.931), phytate intake (P = 0.187) or serum iron (P = 0.373), neither in the anthropometric and hematological indicators.
The simple linear model to evaluate the presence of the parasite A. duodenale, showed a negative significant effect on serum iron (B = −18.2 ± 8.4 µg/dl, r = −0.316, P = 0.035) (
the serum iron in children with A. duodenale is 18.2 µg/dl lower than the level in children who are parasite-free. There was no significant effect on the serum zinc (P = 0.802) (
One way ANOVA analysis for evaluating the effect of polyparasitism showed no significant differences in the parameters of the dietary intake, anthropometric indicators, hematologic and trace elements in children presenting 0, 1, 2, 3 or 4 species of different parasites.
The most striking findings of the present study are the very low levels of serum zinc and iron in the children, the absorption of these minerals from their diet showed to be impaired by phytates content in their diet and by the presence of intestinal parasites.
The low levels of serum zinc can be due to several factors: one is a low intake of zinc, as 30% of the children has inadequate zinc intake. Besides, the main sources of zinc in the studied diet were plant-based foods that provide low bioavailability zinc, due to the presence of phytate, which is mainly found in cereal grains, legumes, seeds and lower levels in tubers [
Furthermore, the serum zinc level of the children was negatively correlated with the phytate intake and with the molar ratio Phy:Zn. A linear regression model showed that serum zinc is decreased by 20.0 µg/dl for every additional unit (g/d) of phytate in the diet with an inversely significant correlation (r = −0.458, P = 0.006). Similar significant inverse correlations (from −0.393 to −0.652 P = 0.05) were reported previously between serum zinc and diets consumed in the same study area [
Intestinal parasitic infections were found to be highly prevalent (96%) in the studied children in this area; results are consistent with those reported previously, showing high parasitic prevalence among rural tropical areas in Bolivia (72 to 100%) [
Therefore, another important factor for the low zinc levels found, is the presence of intestinal parasites. Several studies have reported the detrimental effect of intestinal parasites on the absorption of various nutrients [
The multiple linear regression analysis indicated that both phytate intake and the presence of G. lamblia significantly affect the serum zinc level of the children. Thus, it is important to mention that the high level of phytate impairs the zinc absorption itself, independently of the presence of the parasites, and that this negative effect is exacerbated by the presence of parasites especially G. lamblia. Moreover, due to the intestinal tissue damage caused by G. lamblia, not only zinc absorption, but also other nutrient absorption may be reduced, which may have an effect on weight gain, and is associated with stunting and other health problems in children [
According to the z-scores HAZ, WAZ and BMIAZ, 37% of the children were stunted, 17% wasted and 17% were underweight. One factor contributing to these results, besides the high prevalence of parasites, might be the low energy intake from 3.3 to 6.4MJ/d, which was shown to be inadequate in 43% of the children, similar results of low energy intake were previously reported in rural areas of Bolivia for a population between 4 to 18 years old where the energy intakes were from 2.9 to 6.3 MJ/d [
The evaluation of iron status in the children showed that 84% of them presented lower values than the reference, 66% are iron deficient and 30% of them present anemia. Furthermore, the low levels of hemoglobin and hematocrit are together associated with severe iron deficiency; these levels associated with the high levels of MCV and normal MCHC are an indication of the presence of macrocytic anemia, which is also associated with vitamin B12 and folate deficiency, in the present study 39% and 65% of the children have inadequate intake of these nutrients respectively, moreover, the intake of iron was inadequate in 34% of the children. The obtained results are comparable to previous studies of low serum iron status in children with intestinal parasites [
Regarding the level of lymphocytes, 63% of the children presented low levels of lymphocytes, indicating the presence of lymphopenia found in several infectious diseases [
In conclusion, the low levels of zinc and iron found in children in this study are associated with the low mineral bioavailability and high levels of phytate in the diet, due to the plant-based diet consumed in the area. The low levels of trace elements are also associated with the presence of parasites, particularly G. lamblia and A. duodenale, which have been shown to have a negative effect on the serum zinc and iron respectively. Studies about the relation between nutritional status, trace elements and presence of parasites in children from rural and urban areas are of great importance for developing appropriate nutritional and therapeutic strategies, as well as intervention programs for preventing future adverse effects of untreated intestinal parasite infections.
Financial support from the Swedish International Development Agency (SIDA) is gratefully acknowledged.