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Center for Human Nutrition, Department of International Health, The Johns Hopkins University, Baltimore, MD;
*
Ministry of Health, Zanzibar, Tanzania;
Communicable Diseases Prevention and Control, World Health Organization, Geneva, Switzerland; and
**
Ivo de Carneri Foundation, Milan, Italy
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30 mo, hemoglobin
was related to hookworms but not malaria. In the younger age group,
male sex and recent fever also predicted lower hemoglobin.
2) The three iron indicators were informative in this
population but did not reflect only iron status. Malaria elevated SF in
younger children and TfR and EP in both age groups. Fever elevated SF
in older children and EP in both age groups, but not TfR.
3) Malaria modified the relation of all three indicators
to hemoglobin. The relation of SF to hemoglobin was weak overall, and
absent in malaria-infected children. EP and TfR were strongly
related to hemoglobin, but this relation was attenuated by
malaria.
KEY WORDS: • iron • anemia • malaria • children • helminth infection • nutrition assessment
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For example, the case
fatality rate for children admitted with severe anemia to one hospital
in rural Tanzania was 6.1% (Alonso Gonzalez et al. 2000
).
In contexts such as this, it has been difficult to elucidate the
relative contributions of iron deficiency, malaria, and helminth
infections to anemia (Gillespie and Johnston 1998
) for
several reasons. Randomized trials of highly efficacious interventions
can demonstrate causal relationships with confidence, but relatively
few such trials have been conducted, and very few have reported
attributable risks. Lacking these, the independent contributions of
these underlying factors could be estimated from cross-sectional
data, but large and well-characterized data sets are required
because the underlying factors themselves are related, and their
effects are difficult to separate statistically. Furthermore, the
validity of iron status indicators in these contexts is uncertain
(Hercberg et al. 1987
, Yip and Dallman 1988
). Thus, it has been difficult to categorize children as
iron deficient or iron replete when they are infected with multiple
parasites.
To address some of these issues, we conducted a randomized, placebo-controlled factorial trial of iron supplementation and anthelminthic treatment of preschool children in rural Pemba Island, Zanzibar, Tanzania, where malnutrition, Plasmodium falciparum malaria and geohelminths are highly endemic. We assessed parasitic infections quantitatively, asked mothers to recall morbidity symptoms in the week before assessment, and also measured hemoglobin, erythrocyte protoporphyrin (EP),3 serum ferritin (SF) and serum transferrin receptor (TfR).
We present in this paper the relationships among parasitic infections,
iron status indicators and erythropoiesis before intervention. These
cross-sectional analyses provide evidence about the relation of
anemia and malarial infection, which is not addressed by the
intervention trial. The cross-sectional data may also provide a
truer estimate of the risk of anemia attributable to helminth
infection. Although the causal nature of the association may be
established by the randomized trial, it will underestimate the
magnitude because the intervention does not completely remove the
infection (Albonico et al. 1999
). The
cross-sectional associations between infection and iron status
indicators may also elucidate the extent to which these indicators
relate to iron status or anemia in infected children.
We therefore used multivariate regression methods to address the following questions: 1) Does subclinical infection with P. falciparum, Ascaris lumbricoides, Trichuris trichiura, hookworms or recent fever predict hemoglobin concentration in Zanzibari preschool children? 2) Do EP, SF and TfR provide useful information about iron status in this setting? 3) Does subclinical P. falciparum infection modify the relation of iron indicators to hemoglobin?
We addressed these questions previously in a cross-sectional
analysis of data from Zanzibari school children, and found that malaria
had little influence on SF, EP or hemoglobin concentrations, or their
relations to each other (Stoltzfus et al. 1997a
).
However, we speculated that the answers to the same questions would be
different for preschool children, whose immune response to these
endemic diseases is different from that of older children. The present
analysis allows us to test this hypothesis. In addition, we have added
serum TfR to our series of indicators in this study.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was carried out in Kengeja village, in Pemba Island,
Tanzania. Pemba is the smaller and northern of the two islands that
comprise Zanzibar, located just off the east coast of mainland
Tanzania. Pemba is almost entirely rural, and the population survives
on fishing, farming, and cultivation of cloves and seaweed for export.
The staple foods are cassava and rice. Meat is eaten rarely, and large
fish are relatively expensive. Small fish, legumes and vegetables are
eaten more commonly. P. falciparum malaria is
holoendemic, with year-round transmission. P.
malariae is also present in <5% of infections
(Stoltzfus et al. 1997a
). A peak in malaria parasite
density is observed in July, following the rainy season in April–May
(M. Albonico, unpublished data). Both hookworm species,
Ancylostoma duodenale and Necator
americanus, are transmitted on the island (Albonico et al. 1998
).
In June–July 1996, we performed a census of the entire village to ascertain the number of children, family structure and basic socioeconomic characteristics of each household. Households with preschool children were assigned a household identification number. We then created a database of all children who would be 6–59 mo of age as of September 1, 1996, according to the parent’s report of the child’s age. This yielded a sampling frame of 684 children. Of the 684 children invited to enter the trial, 614 completed the baseline assessment in September 1996. At the time of the baseline assessment, we verified each child’s age using an official document, such as birth certificate or immunization record. Children’s official ages ranged from 4 to 71 mo, and this is the age variable used for analysis.
Data from 490 children are used in these analyses because of the small amount of serum available for assays. After using drops of whole blood for a blood film, and hemoglobin and EP determinations, the remaining serum was collected and assayed for SF and TfR, in that order. Of 614 children assessed at baseline, 124 samples had insufficient serum to complete all of the analyses. Of the 490 children included in these analyses, 23 were missing helminth infection data (see below) and 3 were missing maternal report of recent fever. Thus, in some multivariate models, the sample size is reduced to 464.
This research was approved by ethical review committees of The Johns Hopkins University School of Public Health, the World Health Organization, and the Ministry of Health of Zanzibar.
Assessments.
On the day before a child was scheduled for the baseline assessment, we
visited the home to invite the parents to participate in the trial. If
they gave their informed consent, we asked them to bring a small sample
of their child’s feces to the baseline clinic, and gave them a
container for this purpose. Fecal samples were stained on the same day
and examined within 1 h of staining by the Kato-Katz method
(WHO 1994
). Parents were highly cooperative with fecal
assessment, and helminth egg counts were obtained for all but 23
children in this study sample (95.3%).
At the baseline clinic, any child with a temperature >38.0°C was referred for treatment and rescheduled for assessment on another date. For afebrile children, we asked the mother whether the child had experienced cough, fever, rapid or difficult breathing, liquid stools or bloody stools during the week before the assessment. These questions were asked using local terms for these symptoms, and answers were recorded as yes or no.
Blood (3 mL) was collected by venipuncture into a vacutainer tube with serum separator gel. Drops of whole blood were dispensed immediately to make a blood film, and for determination of hemoglobin using a HemoCue hemoglobinometer (HemoCue, AB, Angelhom, Sweden) and EP using a fluorometer (Aviv Biomedical, Lakewood, NJ). The remaining blood was centrifuged at 1000 x g for 20 min at room temperature and serum was collected.
Thin blood films were fixed with ethanol and stained with Giemsa, and
malaria parasites were counted against leukocytes. The microscopist
counted fields containing >200 leukocytes. If <10 parasites were
seen, the counting continued up to 500 leukocytes. Parasite densities
were calculated by assuming 8 x 109 leukocytes/L
blood (Trape 1985
).
Sera were stored in Pemba at -10°C for up to 3 mo, then transported in liquid nitrogen to Baltimore, MD, where they were stored at -70°C until analysis. Ferritin was assayed using a fluorescence-linked immunoassay (DELFIA System by Wallac, Gaithersburg, MD). The average CV for this assay was 3% (range: 0.2–7.0%). TfR was assayed by ELISA (Ramco, Houston, TX), with an average CV of 4% (range: 0.2–11.8%).
Statistical analysis.
In initial analyses, we did not want to impose any prior expectation on the relation of the iron status indicators to each other or to hemoglobin. Therefore, we built regression models separately for hemoglobin, SF, EP and TfR without including other iron status indicators as independent variables.
Ferritin concentrations were skewed to high values; therefore they were log transformed during model fitting and then reexpressed in their natural units for reporting. Hemoglobin was normally distributed. EP and TfR were somewhat skewed to high values, but the log transformation did not make the distributions Gaussian, nor did it significantly improve the fit of the models; thus we analyzed them in their natural scales.
The following independent variables were considered: malaria parasitemia, Ascaris infection, Trichuris infection, hookworm infection, fever in the past week, sex and age. Malaria parasite densities and helminth fecal egg counts were considered both as dichotomous variables (positive vs. negative) and as categories of infection intensity. Categorical variables were modeled as dummy variables; that is, an ordinal relation was not imposed. Both main effects and interactions between variables were considered. Except for the TfR model, at least one key relation in each model was modified by age; that is, infectious disease had different effects on iron status or erythropoiesis in younger children compared with older children. This statistical interaction was consistently strongest when age was dichotomized using 30 mo as the cutoff. Therefore, we present results separately for these two age groups. Even when modeling the two age groups separately, age in months was always entered into the model and was retained if it remained significant in either age group.
We present the results from these regression models as the adjusted
least-squares means and SEM estimated by the model for
each level of the independent variable. These values may be interpreted
as the adjusted mean value of the dependent variable for a given level
of the independent variable, at the average value for all other
independent variables in the model (i.e., adjusted for other variables
in the model). We tabulated separately the adjusted mean values for
children <30 mo and 30 mo to present the magnitude of the effect
modification by age. The significance level (P-value) of
each age interaction term is given in the footnotes of Tables 2
, 3
and 5
.
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To test whether malaria modified the relation of anemia to iron status indicators, we added hemoglobin and a hemoglobin x malaria interaction term to the multivariate models for SF, TfR and EP. We modeled hemoglobin as a categorical dummy variable, based on the quartiles of the overall distribution, so as not to impose a linear relation between hemoglobin and the dependent variable. We reduced the four malaria parasite density categories used previously to three categories by combining the middle two categories, because of small sample size in some categories and because the middle two categories gave similar results in the models. Thus the malaria x hemoglobin interaction term yielded 12 subgroups, with n ranging from 9 to 95. We examined these malaria x hemoglobin interaction models separately for the younger and older age groups, but present the results for the age groups combined because differences by age were either small or statistically weak (P > 0.05), given the available sample size. These analyses were conducted using SYSTAT statistical software (SPSS, Chicago, IL).
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RESULTS |
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Anemia was highly prevalent and severe, with 80.4% of hemoglobin
concentrations <100 g/L and 15.5% of values <70 g/L. Severe anemia
was strongly concentrated in children <18 mo of age, with 40.2%
having values <70 g/L. Serum TfR and EP concentrations were well above
normal limits, consistent with iron deficiency (Table 1
); however, SF concentrations were not correspondingly low. Only 13.9%
of SF values were <12 µg/L, and the median value was 30.6
µg/L. Hemoglobin, TfR and EP concentrations were strongly
associated with age, improving with age from 4 to 35 mo, and reaching a
stable but still abnormal mean value in children >36 mo of age.
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). Nearly half of the mothers (45.2%) reported that their child
had fever in the week before the clinic visit. Fever was not strongly
related to malaria parasitemia. In children 30 mo, there was no
relation between fever and the presence of parasitemia or parasite
density. However, in children <30 mo, fever was more common in
children with 5000 parasites/µL blood (59.4%
prevalence) than in children with <5000 parasites/µL
blood (35.0%, P = 0.007 by chi-square test). There
was no association between fever and malaria parasitemia below this
threshold.
Infection with each of the three geohelminths increased steeply with
age. Only 5% of children 48 mo were negative for all three
geohelminths. By chance, the prevalence of all three geohelminths in
the youngest age group was identical, representing 6 out of 35 infants
infected with each geohelminth. However, these were not the same 6
infants. Of the 35 infants with fecal smears, 12 were infected with at
least one helminth. Seven were infected with one, four were infected
with two, and one infant was infected with all three helminths. This
last-mentioned infant was an 11-mo-old girl whose hemoglobin, SF,
EP and serum TfR concentrations were 68 g/L, 4.9 µg/L, 395
µmol/mol heme and 16.4 mg/L, respectively.
The three geohelminth infections were significantly associated with each other, even after adjusting for age. In Trichuris-infected vs. noninfected children, the age-adjusted odds ratio for hookworm was 4.7 (95% confidence interval: 2.8–7.9) and for Ascaris was 4.1 (2.5–6.9). Hookworm and Ascaris infection were less strongly associated. The age-adjusted odds ratio for Ascaris infection associated with hookworm infection was 1.9 (1.2–2.9).
Hemoglobin and anemia.
In children <30 mo of age, sex, recent fever and malaria parasite
density were strongly related to hemoglobin concentration (Table 2
). Boys had a hemoglobin deficit of 7 g/L compared with girls. Children
with recent fever had hemoglobin values that were 7 g/L lower on
average than those without recent fever. The strongest predictor of
hemoglobin concentration in this age group was malaria parasite
density. In the lowest and highest categories of malaria parasite
density, mean hemoglobin concentration differed by 19 g/L. All hookworm
infections in this age group were of low intensity.
Hookworm-infected children had average hemoglobin values 4 g/L
higher than children without infection.
In children 30 mo old, there was no sex difference in hemoglobin, and
the difference with recent fever became smaller (3 g/L) and marginally
significant (P = 0.057). Hemoglobin concentrations did
not vary with malaria parasite density, but decreased strongly with
increasing hookworm infection intensity. In this older age group, heavy
hookworm infection intensities (>4000 eggs/g feces) were observed, but
were relatively uncommon (3.5% prevalence). These heavily infected
children had hemoglobin concentrations 19 g/L lower than their
uninfected counterparts. In this age group, the regression model
explained only 9% of the variation in hemoglobin (multiple
R2 = 0.091).
The different effects of malaria and hookworm infections on hemoglobin
in the two age groups were also apparent when severe anemia (hemoglobin
<80 g/L) was considered as the outcome (Fig. 1
).
|
With EP and TfR in the model for younger children, the effect of hookworm infection became small and nonsignificant. The effects of sex, fever and malaria were attenuated but remained strong. Adding EP and TfR to the model for older children attenuated but did not remove the effect of hookworm.
Serum ferritin model.
In children <30 mo old, SF concentrations increased directly with
malaria parasite density (Table 3
). In children 30 mo old, SF was not associated with malaria parasite
density, but decreased strongly with increasing intensity of hookworm
infection. SF concentration was also higher in children with recent
fever in this age group.
Trichuris infection intensity had a similar nonsignificant
(P = 0.20) pattern of association with SF
concentration in both age groups. Ferritin concentrations were slightly
elevated in children with light infections and much lower in the few
children with 5000 eggs/g feces. This relation was significant
(P = 0.041) when the two age groups were combined.
Serum transferrin receptor.
After adjusting for age, the only significant relation between serum
TfR and infection was with malaria (Table 4
), and this association was similar in older and younger children in the
study. TfR concentration increased directly with level of malaria
parasite density, but the difference was greatest between
malaria-negative children and malaria-positive children (mean
± SEM: 12.0 ± 0.9 vs. 14.7 ± 0.4 mg/L,
P = 0.007).
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EP to heme ratios were higher in children with recent fever and were
also higher in children with higher malaria parasite densities
(Table 5
). The association with fever was of similar magnitude in younger and
older children, and significant (P = 0.003) when the
ages were combined. The increase in EP with malaria parasite density
was marginally significant in each age subgroup, and statistically
stronger when the ages were combined (P = 0.021). It
appears that the relation might have a different threshold in younger
vs. older children in our study. In children <30 mo old, EP values
were elevated in children with any parasites present, but in children
30 mo old, EP values were elevated only in children with 5000
parasites/µL blood.
EP was associated with hookworm infection in both age groups, but
in opposite directions (Table 5)
. In children <30 mo old, those with
light hookworm infections had lower EP values, with a difference of 67
µmol/mol heme. Moderate and heavy hookworm infections were
not found in this age group. In children 30 mo old, EP values were
significantly higher in children with 4000 hookworm eggs/g feces
compared with children with egg counts below this threshold (mean
± SEM: 155 ± 17 vs. 219 ± 30
µmol/mol heme, P = 0.034). The reverse
association in children with light infection compared with no
infection, observed in younger children, was not found in the older age
group.
Malaria as an effect modifier.
In the SF model (Fig. 2A
), the interaction term for hemoglobin and malaria was nonsignificant
(P = 0.194), and the model fit poorly when the
hemoglobin interaction was added (multiple
R2 = 0.082). SF was related much more
strongly to malaria parasite density than it was to hemoglobin.
However, in children with a negative blood film for malaria, SF was
significantly lower in the lower hemoglobin quartiles than in the upper
two quartiles (adjusted geometric mean SF 14.7 vs. 27.0
µg/L, P = 0.014). Motivated by the strong
age modification of SF predictors in Table 3
, we examined further the
relationship between SF and hemoglobin in children negative for
malaria, by age group. The positive association between hemoglobin
quartile and SF derived entirely from children <30 mo of age
(n = 50 without malaria, P = 0.055),
and was absent in children 30 mo of age (n = 30
without malaria, P = 0.60). The absence of a
relationship in older children might be due to the strong influence of
fever in this age group (see Table 3
).
|
). EP was strongly related to hemoglobin, regardless of
malaria parasite density (Fig. 2B
). However, this relation
was strongest in children with negative blood films. Malaria modified
the relation between hemoglobin and EP, and this modification was
different in the lowest hemoglobin quartile than in the upper three
quartiles. In children with hemoglobin <76 g/L (lowest quartile), EP
values in malaria-infected children (regardless of parasite
density) were much lower than those in malaria-negative children.
In the upper three hemoglobin quartiles, EP values were not affected by
malaria parasitemia unless it exceeded 5000 parasites/µL
blood. In these three hemoglobin quartiles, EP values of children with
high parasitemia were significantly elevated and rose more steeply with
descending hemoglobin level [malaria (5000 parasites) x hemoglobin interaction term in upper three quartiles, P
= 0.125].
In the TfR model (Fig. 2C
), both hemoglobin and its
interaction term with malaria were significant, and this interaction
model fit the data much better (multiple
R2 = 0.219) than the simpler model in
Table 4
. In children with negative blood films, TfR concentration
descended steeply with ascending hemoglobin level. In children with low
malaria parasite densities, this relation remained strong, but was
attenuated. In children with 5000 malaria parasites/µL
blood, the relation was U-shaped.
The subgroup of children with hemoglobin 98 g/L (highest quartile)
and malaria parasite density 5000 was small (n = 9);
their adjusted mean TfR value plotted in Figure 2C
(21.5
mg/L, range 8.1–51.3) was influenced by two very high values (case A,
48.4 mg/L, and case B, 51.3 mg/L). Case A and case B had malaria
parasitemias of 5066 and 5134 parasites/µL blood,
identical hemoglobin concentrations of 99 g/L, and EP values of 134 and
73 µmol/mol heme, respectively. To reduce the influence of
these two points, we examined the malaria x hemoglobin
interaction using the median values of TfR rather than the
age-adjusted geometric mean. Using median values, the interaction
was less striking, but did not disappear. Median TfR values for
ascending hemoglobin quartiles were 18.1, 13.0, 9.9 and 8.6 mg/L for
malaria-negative children, and 16.4, 14.8, 12.2 and 13.0 mg/L for
children with 5000 parasites/µL blood.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), intense
transmission of P. falciparum infection in young children
alters the metabolism of children who are not ill in remarkable ways. Epidemiology of anemia.
Our observation that malaria parasitemia is associated with lower
hemoglobin concentration in young children confirms earlier
observations of Spencer (1966)
in Papua New
Guinea, and MacGregor et al. (1966)
in The Gambia, who
also observed that the hemoglobin deficit associated with parasitemia
decreased with age. In the Pemban children we studied, this relation
was absent beyond the age of 30 mo. There was also no significant
association of malarial infection and hemoglobin concentration in our
previous study of Pemban school children (Stoltzfus et al. 1997a
). This is consistent with the development of
partial immunity to malaria that is acquired rapidly in areas of very
high transmission (Snow et al. 1999
).
In these young Pemban children, both hemoglobin and risk of severe
anemia were strongly and directly related to the malaria parasite
density. It is possible that higher parasite density increases the rate
of hemolysis, resulting in a more severe anemia in children who cannot
produce new red cells at an equally accelerated rate. Erythropoiesis
might be limited by folate deficiency, iron deficiency, insufficient
erythropoietin production or cytokine-mediated suppression of the
marrow response to erythropoietin. A greater population of circulating
parasites might also cause more iron to be incorporated into hemozoin
complexes (Brabin 1992
).
All three geohelminths were prevalent in the children studied, but only
hookworm was associated with hemoglobin or severe anemia. In children
30 mo old, these associations were consistent with the
well-described intensity-dependent relation between hookworm
infection and intestinal blood loss (Roche and Layrisse 1966
, Stoltzfus et al. 1996
). The hemoglobin
deficits associated with hookworm infection in this age group were
related to iron deficiency. This was evidenced by the relation of both
SF and EP to hookworm infection intensity, and the fact that
hookworm’s association with hemoglobin was attenuated when iron status
indicators were added to the hemoglobin model.
In children <30 mo old, hookworm-infected children had
significantly higher hemoglobin concentrations, less severe anemia and
lower EP concentrations than uninfected children. Because these were
light hookworm infections and, given the young age group, relatively
recently acquired infections, it is not surprising that hookworm
infection was not associated with iron-deficiency anemia
(Roche and Layrisse 1966
). It is surprising, however,
that hookworm-infected children in this age group had significantly
better hematologic status. Hookworm infection is transmitted
through direct contact of the skin with larvae-infected soil (in
contrast to Ascaris and Trichuris infections,
which are transmitted orally). It is possible that young children with
severe iron-deficiency anemia are less active or exploratory in
their behavior (de Andraca et al. 1997
) and are
therefore less likely to acquire infection at a very young age.
In children <30 mo old, boys had a sizeable hemoglobin deficit (7 g/L)
relative to girls. This was apparently due to poorer iron status and/or
erythropoiesis in the boys, because sex did not remain an important
predictor of hemoglobin in the multivariate model after EP and TfR were
added. The explanation for this is not clear, and it might be due to
chance. However, in our study of Zanzibari school children we also
found that boys had a small hemoglobin deficit relative to girls
(Stoltzfus et al. 1997b
).
In the younger age group, children recovering from recent fever were
more anemic. Although we adjusted for current malaria parasite density,
it is possible that the effect of fever reflects past malarial illness
(at least in part) because fever in the model occurred in the week
before the day the blood film was made. In our previous study of school
children, fever in the past week was also associated with a small
hemoglobin deficit (117 vs. 115 g/L, P = 0.005,
n = 3298, R. J. Stoltzfus, unpublished data),
similar to the present observation in older preschool children. We
speculate that certain immunologic responses (e.g., secretion of tumor
necrosis factor-
) underlie the effect of fever on erythropoiesis and
that these responses change with normal child development, creating a
strong age modification of fever’s effect.
Serum ferritin, erythrocyte protoporphyrin and serum transferrin receptor.
SF, EP and TfR are iron status indicators, but each reflects a particular physiologic process. It is important to evaluate how these assessments performed as indicators of iron status in this population, but the present cross-sectional analysis can provide only limited answers. The most informative question may not be "Were they accurate indicators of iron status?" but rather, "What do they tell us about iron metabolism and erythropoiesis in these children?"
SF is an iron storage protein that is also an acute phase protein
(Bentley and Williams 1974
, Cook et al. 1974
, Elin et al. 1977
). Both of these
properties are evident in the data. The association of SF and recent
fever reflects the well-known acute phase response. The strongly
age-dependent relation of SF to malaria parasite density mirrors
the hemoglobin-parasitemia relationship in these children. It
appears that the age-dependent immune mechanisms that protect older
children from the anemia of subclinical malarial infection might also
remove the SF response to malaria infection. This supports our previous
hypothesis that the absence of association of SF and malarial infection
in Pemban school children was an age- and immunity-dependent
observation (Stoltzfus et al. 1997a
).
The malaria-SF relationship disappeared with age, whereas the
influence of fever became stronger with age. Indeed, the strongest
relationship of SF to hemoglobin was found in the subgroup of children
who were malaria-free and <30 mo of age. From this, we infer that
the effect of inflammation on SF does not disappear with age; rather,
the effect of subclinical malarial infection on inflammation (and
therefore SF as an acute phase protein) disappears with age in
holoendemic populations. This is consistent with the fact that in
Zanzibari school children, malarial infection was not associated with a
significant elevation in SF (Stoltzfus et al. 1997a
),
but recent fever was associated with SF (geometric mean SF in 422
school children who reported fever in the past week, 19.8 vs. 16.9
µg/L in 2606 children who did not report recent fever,
P < 0.001, R. J. Stoltzfus, unpublished data).
Even in the face of its behavior as an acute phase reactant, SF does
carry information about iron status. This is apparent in the strong
relation between hookworm infection intensity and SF concentration in
the older age group. Hookworm infection, even in children as young as
2–5 y old, causes intestinal blood loss sufficient to deplete iron
stores. It is possible that the nonlinear relation of
Trichuris infection intensity and SF reflects both
properties of SF. At low infection intensities, which are likely first
infections in this age group, SF is elevated, possibly reflecting an
inflammatory response to Trichuris in these nonimmune
children. At high infection intensities, SF concentrations were very
much lower than in the population overall, possibly reflecting
intestinal iron loss (Layrisse et al. 1967
).
In children with no malaria parasites, SF was significantly higher in
those with hemoglobin concentrations above the median value (Fig. 2A
), which again reflects ferritin’s role as an iron status
indicator. However, the shape of the relation was not consistent with
solely iron-deficiency anemia. At hemoglobin concentrations <88
g/L, SF concentrations were low (geometric mean 14.7 µg/L)
but not as low as expected for this severity of iron-deficiency
anemia. This suggests that inflammatory responses in these anemic
children were marginally elevating SF, or that the children were not
uniformly iron deficient, despite their very low hemoglobin levels. In
the upper two hemoglobin quartiles, ferritin concentrations were
higher, but did not demonstrate the expected increasing values with
increasing hemoglobin. Again, this suggests that ferritin’s behavior
as an acute phase reactant is obscuring the true relation of hemoglobin
to iron stores, or that a large part of the mild-to-moderate anemia in
this population is not due to iron deficiency.
EP accumulates during iron-deficient erythropoiesis. Zinc
protoporphyrin IX, the form that we measured, is produced when iron is
not available for the conversion of protoporphyrin to heme. When the
iron supply is adequate, EP is not elevated, even after experimental
phlebotomy in humans that reduced their hematocrit to 55% of its
original level and increased their reticulocyte counts to 2–3 times
normal (Langer et al. 1972
). When healthy rabbits were
phlebotomized to increase their reticulocyte counts to 7 times normal,
EP values became elevated (2.6 times normal) unless the rabbits were
transfused with nonviable erythrocytes to provide an additional supply
of iron (Langer et al. 1972
). Thus EP remains specific
to iron deficiency even in the face of significant erythropoiesis.
Therefore, we interpret the inverse relation between hemoglobin and EP in this population to be strong evidence of iron-deficiency anemia. Indeed the association of EP to hemoglobin was stronger than that of serum TfR or SF (which had no association with hemoglobin). The high EP values in children with heavy hookworm infections is evidence for iron-deficient erythropoiesis in those children, as expected from their SF values and from prior knowledge of hookworm-related blood loss.
EP was higher in children with recent fever and in children with
malaria parasitemia. Elevations in EP measured by the fluorometric
method we used have been reported in patients hospitalized with a
variety of diseases, due to substances in the plasma that fluoresce at
the same wavelength as zinc protoporphyrin IX (Hastka et al. 1992
). These substances can be removed by washing the red cells
before measuring the EP (Hastka et al. 1992
), a
time-consuming process that we did not do. Although the children in
our study were not acutely ill, it is possible that those with recent
fever had some plasma metabolites that elevated our EP measurements.
Malaria parasitemia modified the relation of EP to hemoglobin in a
complex way. At hemoglobin values 76 g/L, EP values were elevated
only in those children with high parasite densities (who comprised 10%
of the sample in that hemoglobin range). Schneider et al. (1993)
also reported elevated EP, measured by the Aviv
hematofluorometer, in young children from Togo with P.
falciparum infection. They hypothesized that bile pigments, which
fluoresce in this assay, are released into the plasma from parasitized
red cells as they are hemolyzed. This might also explain our finding.
At hemoglobin <76 g/L, all children with circulating parasites had
markedly lower EP values than malaria-negative children. Zinc
protoporphyrin IX is toxic to the malaria parasite through a mechanism
that appears to be identical to many antimalarial drugs (Iyer 1999
, Martiney et al. 1996
), and we hypothesize
that children who accumulate very high levels of EP may be protected
from parasitemia.
TfR is a surface receptor expressed by nearly all human cells, but 80%
of the serum receptor concentration comes from erythroid cells
(Huebers et al. 1990
). The concentration of serum TfR is
elevated by iron deficiency and by erythropoiesis (Kling et al. 1998
). TfR was first proposed as a simple marker of
erythropoiesis (Kohgo et al. 1987
). Serum TfR is not an
acute phase protein and has therefore been proposed as a promising
indicator of iron status in populations with prevalent infections
(Ferguson et al. 1992
), including malarial infection
(Kuvibidila et al. 1995
). In our data, serum TfR was the
only iron status indicator that was not altered in children with recent
fever.
We found that malaria-infected children had higher concentrations
of serum TfR and that malaria parasitemia modified the expected
relation to hemoglobin. We hypothesize that serum TfR in this
population of children was reflecting mainly erythropoiesis. According
to this hypothesis, children with higher malaria parasite densities
experience hemolysis, which reduces the red cell mass and stimulates
erythropoiesis. Thus, elevated serum TfR concentration would indicate a
physiologic response to the anemia of malaria. Our observation that
serum TfR was lower in severely anemic children with malaria
parasitemia than in severely anemic children without malarial infection
may reflect a suppression of erythropoiesis caused by malaria. This
might be caused by suppressed erythropoietin secretion in the anemia of
malaria (Vedovato et al. 1999
) or by some
malaria-induced blockade of the marrow response to erythropoietin
(Yap and Stevenson 1994
). In either case, the erythroid
response to severe anemia would be attenuated, as we observed.
Kuvibidila et al. (1995)
reported normal serum TfR
concentrations (mean value, 5.0 mg/L) in 17 Zairian children with
symptomatic malaria, despite a mean hemoglobin concentration of 61 g/L.
In the presence of such severe anemia, this lack of erythropoiesis is
pathological (Beguin et al. 1993
), and consistent with
simultaneous hemolysis and suppressed erythropoiesis in acute malaria.
In the asymptomatic severely anemic children we assessed,
erythropoiesis (as reflected by serum TfR) was suppressed in
malaria-infected compared with noninfected children, but was still
significantly elevated (mean serum TfR, 17.5 mg/L, compared with normal
values of 2.9–8.3 mg/L published using the Ramco ELISA kit
(Yeung et al. 1998
).
Serum TfR concentrations were also elevated in children with high
malaria parasite densities but hemoglobin concentrations >98 g/L.
These children were rare (9 of 490), and are remarkable for their
capacity to maintain relatively high hemoglobin levels despite high
parasitemia. It is possible that these children are compensating
effectively for hemolysis by producing new red cells at a faster rate
than their more anemic counterparts, perhaps because their cytokine
response to malaria is less suppressive to the bone marrow, or because
their iron or folate status is sufficient to maintain a high level of
erythropoiesis. The relatively low EP values in these 9 children (see
Fig. 2B
) are consistent with a relatively permissive iron
supply. In future studies, it would be informative to assess
reticulocyte counts together with serum TfR in a population such as
this to confirm the extent to which serum TfR is reflecting
erythropoiesis (Beguin et al. 1993
).
We draw the following conclusions with regard to our three research
questions. 1) Low hemoglobin was related most strongly to
malaria parasite density in children <30 mo, and to hookworms in
children 30 mo. In the younger age group, male sex and recent fever
also predicted lower hemoglobin. We conclude from the strong relation
of EP to hemoglobin that iron deficiency was also an important cause of
low hemoglobin in this population.
2) All three indicators were informative in this population, but they did not reflect only iron status. Malaria infection significantly influenced all three indicators, and fever influenced EP and SF. Even with this reasonably large and well-characterized data set that includes multiple indicators of iron status, it would be nearly impossible to conclude which children were iron deficient or to estimate the absolute prevalence of iron deficiency in this population. The only way to determine the fraction of anemia attributable to iron deficiency in this situation is to conduct a highly supervised course of therapeutic iron supplementation. Nonetheless, each indicator we assessed provided unique information about children’s hematologic status.
3) Malaria modified the relation of all three indicators to hemoglobin. This suggests that malaria not only shifts the absolute values of the indicators, but that the indicators reflect different processes in malaria-infected children than in normal children. This effect was especially large and significant in the case of TfR. The effects of subclinical infection on erythropoiesis in young children deserve further study to elucidate both the etiologies of anemia and the best methods for iron status assessment. The interpretation of serum TfR in young children experiencing intense malarial transmission is a particularly salient question.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: EP, erythrocyte protoporphyrin; SF, serum ferritin; TfR, transferrin receptor.
Manuscript received November 19, 1999. Initial review completed January 26, 2000. Revision accepted March 7, 2000.
|
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