Relation of Nutritional Status, Sickle Cell Trait, Glucose-6-Phosphate Dehydrogenase Deficiency, Iron Deficiency and Asymptomatic Malaria Infection in the Niger Delta, Nigeria
Zaccheaus A. Jeremiah,
Emmanuel K. Uko
Essien A. Usanga
This study aimed at investigating these complex interactions with a view
to ascertaining the risk or benefit of acquiring these factors in a malaria
endemic part of Nigeria. In a cross sectional study, 240 asymptomatic,
non-hospitalized children aged 1-8 years of both sexes were assessed for
nutritional status (using anthropometric indexes), malaria parasite, hemoglobin,
white blood cell count, hemoglobin electrophoretic pattern, glucose-6-phosphate
dehydrogenase deficiency and serum ferritin concentrations. Fifteen percent
of the children were malnourished (BMI Z score <-2), 12.5% were iron
deficient (serum ferritin <12 ng mL-1), 12.5% were HbAS,
87.5% HbAA while 5% of the children were G6PD deficient. The mean parasite
density of the study population was 1.14 x 10 3 parasites μL-1
and the risk of malaria infection was higher in iron replete children
than those who were iron deficient (RRR = 0.33, χ 2 = 2.825,
p<0.05). The risk was age-related, higher in the under fives than the
5-8 years group (RRR =1.7, χ 2 = 2.910; p<0.02). There
was an association between low serum iron concentration and sickle cell
trait (χ 2 = 35.890; p = 0.056), no statistical significant
risk was observed with nutritional status, hemoglobinopathy and G6PD deficiency.
This study provides an observational support that iron deficient children
are to some extent protected against malaria infection and malnutrition
places children below five years of age at risk of malaria related morbidity.
to cite this article:
Zaccheaus A. Jeremiah, Emmanuel K. Uko and Essien A. Usanga, 2008. Relation of Nutritional Status, Sickle Cell Trait, Glucose-6-Phosphate Dehydrogenase Deficiency, Iron Deficiency and Asymptomatic Malaria Infection in the Niger Delta, Nigeria. Journal of Medical Sciences, 8: 269-274.
A large percentage of child deaths related to malaria are attributable
to under nutrition and deficiencies of vitamin A, zinc, iron and folate
(Steketee, 2003) Nutritional status strongly influences the disease burden
of malaria, however this relationship remains unclear and controversial
(Nyakeriga et al., 2004). Many individuals at risk of malaria have
micronutrient deficiencies that may hamper protective immunity. Nutrition
appears to influence susceptibility to malaria and affects the course
of the infection (Shankar, 2000). Published data from developing countries
indicate that children generally fail to achieve their genetically determined
potential growth because of poor diet and infection (Ulijaszek, 1994;
Waterlow, 1994). On the other hand, several studies have suggested that
malnourished children are protected to some degree against malaria (Walker
et al., 1996; Nyakeriga, 2005).
In malaria endemic areas, host factors especially the genetic red cell
disorders, including sickle cell trait and enzyme deficiencies (glucose-6-phosphate
dehydrogenase deficiency) have been shown to confer natural protection
against malaria infection hence the term natural immunity. All these factors
affect parasite survival and provide resistance in malaria endemic areas,
the so called malaria hypothesis (Berkley et al., 1999; Newton
et al., 2000).
Glucose-6-phosphate dehydrogenase is an enzyme in the hexose monophosphate
shunt responsible for the generation of reduced glutathione. This reduced
glutathione protects sulphydryl group of hemoglobin and the red cell membrane
from oxidation by the oxygen radicals. Defects in the shunt leads to inadequate
protection against oxidation, resulting in oxidation of sulfhydryl groups
and precipitation of haemoglobin as Heinz bodies and in lysis of the red
cell membrane (Frank, 2005).
In order to elucidate the complex web of interactions between nutritional
status, sickle cell trait and G6PD deficiency, a study was undertaken
in a malaria endemic part of the Niger Delta region of Nigeria. The study
aimed at investigating the relationship between iron status, sickle cell
trait, G6PD deficiency and malaria with a view to ascertaining the risk
or benefit of acquiring these conditions in a malaria endemic area.
MATERIALS AND METHODS
Study area and population: This study was conducted between March
2005 and April 2006 in Rumueme, Port Harcourt. The geographical location
is latitude 4° 31`-5° 31`and longitude 6° 30`-7° 21`.
The typical deltaic wetlands and mangrove forests that climatize the area
provides enough breeding grounds for mosquito and malaria transmission
throughout the year, although the majority of clinically evident infections
occur after long and short periods of rainfall, that in general occur
during months of October- November and March to July every year, respectively.
The study population consisted of 240 children (1-8 years) of both sexes
recruited from households and schools.
A cross sectional study design was used in this study. Eligibility criteria
for this study were as follows; (1) axillary temperature of ≤37.5°C,
(2) age 1-8 years, (3) absence of symptoms suggestive of malaria or any
other systemic illness (4) parental consent.
This study received ethical approval form the Rivers State University
of Science and Technology and informed consent was received from each
parent before samples were collected for analysis. All the children were
weighed using the YAMATO digital scale (China). Heights were measured
in children ≥1 year with a Leicester height measure (MS instruments).
The temperature of each child at the time of blood collection was taken
using DGL digital thermometer (MODE ECT- 1, Germany).
Collection of blood samples: Four milliliter of whole blood was
drawn with syringe (5 mL) through venepuncture using the antecubital vein
and dispensed into an ethylenediamine tetracetic acid (EDTA) and was used
for malaria parasite estimation, hemoglobin electrophoresis and glucose-6-phosphate
dehydrogenase deficiency while the remaining two milliliter in the syringe
was used for serum ferritin concentration determination.
Methods: Malaria was estimated by microscopy with well stained
Giemsa smears (thick and thin) using 100x oil immersion. Thick and thin
blood smears were stained by fresh working Giemsa stain according to standard
procedure. Parasite densities were recorded as a ratio of parasites to
white blood cells (500 WBCs) from thick smears. Densities (parasites μL-1)
= parasites/500 WBCs x WBC count of individual subjects.
White blood cell count was done by diluting a well mixed whole blood
with Turk`s solution in the ratio of 1 in 20 (950 μL of Turk`s solution
to 50 μL of whole blood. The haemocytometer was then filled with
an aliquot of this mixture and allowed to settle for one minute prior
to performing the count, white blood cells were counted with x10 objective
with reduced light in the four corner squares of the counting chamber.
The member of white blood cells/cubic milliliter was calculated as follows:
cells counted x dilution factor x chamber depth/area of chamber counted.
Haemoglobin electrophoretic pattern of the participants were determined
using haemoglobin electrophoresis by cellulose acetate membrane at pH
8.9 as described by Brown (1993). A small quantity of haemolysate of venous
blood from each of the subjects was placed on the cellulose acetate membrane
and carefully introduced into the electrophoretic tank containing Tris-EDTA-Borate
buffer at pH 8.9. The electrophoresis was then allowed to run for 15-20
min at an electromotive force of 160 V. The results were read immediately.
Haemolysates from blood sample of known haemoglobin (i.e., AA, AS, AC)
were included as controls.
A qualitative determination of G6PD in red cells of participants was
carried out using the G6PD deficiency screen reagent set (Pointe scientific,
USA. Lot # 513704). A red cell haemolysate was prepared by adding 50 μL
of whole blood to 2.5 mL deionised water, mixed gently and allowed to
stand for 5 min. Into 13x100 mm test tubes labeled positive, negative
controls and samples were dropped 500 μL of reconstituted G6PD screen
reagent. 1.0 mL of the haemolysate was then added into each tube and gently
shaken to mix. One milliliter of mineral oil was then gently layered on
top of the reaction mixture. The tubes were observed at 15 min interval
for up to 1 h for colour change from deep purple to red/orange, Negative
samples did not show any colour change.
Nutritional status of the children were determined using anthropometrical
indexes. Z-scores were determined using the CDC Epi Info V 6.04 to classify
children as undernourished (BMI Z <- 2), well nourished (BMI Z-0.5
to 2), overweight (BM Z>2), Haemoglobin level of participants were
estimated using the cyanmehaemoglobin method with reagents bought from
Pointe Scientific Inc, USA (Catalog #H 7504-500).
Statistics: Data were arranged in a 2x2 contigency table and analyzed
using the statistical package for social science (SPSS) (Version 11.0,
Chicago, IL.USA). Descriptive statistics of continuous variables were
expressed as Mean±Standard deviation. The students t-test was used
for the comparison of means. Relative Risk Ratio (RRR) and Chi-square
values were calculated using standard formulae. Non parametric test (Kruskal-Wallis)
was employed for the analysis of skewed data. Significant level was set
Only the temperature and haemoglobin values showed significant difference
between the parasitized and non-parasitized children (p<0.02 and p<0.01,
respectively) (Table 1).
Table 2 shows the nutritional status of the children
based on BMI Z scores. 15% were undernourished, 13.75% at risk of under
nutrition, 35% well nourished and 36.25% overweight. There was no significant
relationship existing between haemoglobinopathy, iron status and malaria
as shown in Table 3. The risk of malaria infection was
higher in iron replete children than those who were iron deficient and
this was statistically significant (RRR = 0.33; χ2 = 2.825;
p<0.05). The risk was also higher in the under fives than the 5-8 years
children (RRR = 1.7; χ2 = 2.910; p<0.02). Malnutrition,
haemoglobinopathy and G6PD deficiency were not found to exert any significant
risk for acquiring malaria infection (Table 4).
||Characteristics of study participants
|BMI: Body Mass Index, WBC: White Blood Cell, *Statistically
significant, ns: Not Significant
||BMI Z scores of 240 children aged 1-8 years in relation
to age groups and
BMI Z-scores Z-scores <-2: Undernourished, Z-scores
<- 1 to 1 at risk of under nutrition, Z scores-0.5 to 2 = Well
nourished, Z-scores > 2 = Over weight
||Relationship between iron status, sickle cell trait
and malaria Hb electrophoretic pattern
|Chi-square (χ2) test = 0.378; p = 0.984ns,
Pearson correlation (R) = 0.028; p = 0.803ns
||Risk analysis of malaria parasite infection in relation
to iron deficiency, nutritional status, sickle cell trait and G6PD
|RRR: Relative Risk Ratio
The prevalence of sickle cell trait in present study population
was 12.5%. Given the high prevalence of asymptomatic malaria and iron
deficiency in our study population (Jeremiah et al., 2007, 2008),
we wanted to test the hypothesis that sickle cell trait protect individuals
from becoming iron deficient and malaria infection. Subsequently, the
association between biochemical indices of iron status and these sickle
cell traits were investigated. Further, we found that iron deficiency
was associated with age, being more prevalent in younger children. Overall,
our results showed that children with sickle cell trait were not protected
from being iron deficient (χ2 = 0.378, p = 0.984). Instead,
we observed an association between low serum iron concentration and sickle
cell trait (χ2 = 35.890, p = 0.056). These observations
are discussed in the light of previous reports. Iron deficiency and malaria
are singly or in combination the most common causes of anemia. However,
the aetiology of malaria is multifactorial, involving host factors such
as haemoglobinopathies (sickle cell trait). The interaction between haeoglobinopoathy,
iron status and malaria is complex and rather controversial. While a large
body of evidence from the literature shows that milder sickle cell trait
is associated with protection against malaria, the sever forms of haemoglobinopathy
have been shown on the other hand to be associated with anaemia and on
the other hand protection against iron deficiency through increased iron
absorption (Hershko et al., 1982).
There is no consensus concerning the specific mechanism of protection
against malaria infection by any of the haemoglobinopathies, but variant
haemoglobin has been associated with various pathophysiological condition.
For example, HbS is associated with auto-oxidation of haemoglobin and
β-thalassemia with ineffective erythropoiesis (Schrier, 2002). Nevertheless,
several mechanisms have been postulated including reduced invasion of
variant red cells by the parasite (HbE, HbC and HbH and/or consequent
reduced growth, reduced cytoadherence of infected red blood cells as well
as reduced rosetting of uninfected red cells and increased clearance of
the infected red cell via phagocytosis. (Roberts and Williams, 2003).
Another possible mechanism through which haemoglobinopathy protection
could protect against malaria is through iron deficiency. While the role
of iron in protection against malaria remains controversial, there is
some evidence that iron deficiency might protect against malaria infection.
In line with this, it has been reported that iron supplementation of pregnant
women with sickle cell trait (HbAS) increased their susceptibility to
malaria (Menendez et al., 1995). This implies that iron deficiency
might contribute to protection against malaria seen in HbAS condition.
In this study there was an association between low serum iron and sickle
cell trait. We speculate that these observations if corroborated by future
longitudinal studies, iron deficiency might be yet another mechanism through
which sickle cell trait might protect against malaria. There was a reduced
risk of malaria infection in the iron deficient children as compared to
the iron replete children (RRR = 0.33, χ2 = 2. 825, p>0.05)
suggesting that iron deficiency was associated with protection against
malaria to an extent.
The relationship between malnutrition and malaria was also examined in
this study. The prevalence of under nutrition (defined as BMI Z scores
<-2) was observed to be 15.0%. When two groups (undernourished) and
well nourished group were subjected to risk analysis, it was observed
that there was no significant difference (RRR = 1.03, χ2
= 0.32 p>0.05) The effect of nutritional status on malaria became manifest
only when age was introduced into the analysis (RRR = 1.7, χ2
= 2.910, p<0.02). Malnutrition and malnutrition associated adverse
effects cuts across all ages. However, in malaria endemic areas, the greatest
impact is seen in the younger children, less than the age of five years
(Snow et al., 1999) Recent estimates combining prevalence data
and the population attributable risk factors revealed that most malaria
deaths were attributed to under nutrition in children less than five years
of age (Caulfield et al., 2004). This study corroborates other
reports in Nigeria that malnutrition and malaria are two twin factors
that are responsible for most infant mortality rates. (Ezedinachi and
Ejezie, 1990; Ejezie and Ezedinachi, 1992; Ekanem et al., 1994;
Olanrewaju and Johnson, 2001).
With respect to glucose-6-phosphate dehydrogenase deficiency (G6PD),
this study observed 5% deficient children and 95% normal G6PD. The distribution
of this genetic trait among the parasitized and non-parasitized group
was not significant. No association either was established with malaria.
The G6PD deficient cells lack the ability to resist sustained oxidative
stress adequately and hence the free radical producing parasite is a challenge
to such cells. This situation is thought to make them more susceptible
to phagocytosis. Also, oxidative stress induced by the parasite plus the
normal red cell oxidative stress particularly unquenched by the enzyme
deficiency, results in a environment in which normal parasite growth is
limited (Nagel, 2004).
A study conducted in Mali by Guindo et al. (2007) among a population
of children with severe malaria indicated a population of children with
severe malaria indicated a protective effect of the G6PD A- allele in
heterozygous females and in males hemizygous for the wild type allele.
This finding was also similar to the report of Ntoumi et al. (2003)
who concluded that G6PD A- heterozygous females are protected against
all forms of Plasmodium falciparum malaria.
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