Iron deficiency is probably the most common form of malnutrition in the world, affecting more than half of the women and children in developing countries (DeMaeyer and Adiels-Tegman 1985). In many regions where iron deficiency is of great public health importance, malaria is endemic and the majority of the population harbors subclinical infections. In Africa alone, an estimated 275 million people are infected with this parasite (Najera et al. 1993). Malaria and other infections have complex effects on iron metabolism that may affect the interpretation of hemoglobin, erythrocyte protoporphyrin (EP) and serum ferritin (Baynes et al. 1986, Brabin 1992, Hastka et al. 1993), commonly used indicators of iron deficiency. Although these effects have been described in studies of individuals acutely ill with malaria (Adelekan and Thurnham 1990, Ayatse and Ekanem 1994, Phillips et al. 1986), there is a need for practical guidance regarding the use of indicators to assess iron deficiency in apparently healthy people in malaria-endemic environments.

The spectrum of iron status covers several well-defined stages, ranging from adequate iron storage to depleted iron stores, iron-deficient erythropoiesis, and iron deficiency anemia (Bothwell et al. 1979). To characterize the iron status of a population or to monitor changes in status, it is desirable to use a combination of indicators that provides information about this entire spectrum. Serum ferritin is a particularly useful indicator of iron status, because it is linearly related to iron stores when stores are present (Cook et al. 1974). Erythrocyte protoporphyrin is a precursor to heme in red cell production. Elevated EP is a sensitive indicator of iron-deficient erythropoiesis (Langer et al. 1972), because EP accumulates when iron is limiting. Hemoglobin concentration is used to define anemia, the end stage of iron deficiency (Anonymous 1989).

The effect of malaria on iron metabolism is not fully understood, but distinct changes in iron metabolism occur during malarial infection that may affect iron status indicators and their interrelationships (Table 1). Malarial disease causes destruction of red blood cells while suppressing erythropoiesis, resulting in a profound anemia (Phillips et al. 1986). These processes would shift iron out of heme toward storage forms. As long as heme iron from destroyed erythrocytes is effectively recycled, these processes would not alter total body stores of iron but would certainly alter the expected relationships among iron status indicators. Malaria may also cause loss of functional body iron, through immobilization in the form of hemazoin (malarial pigments) and through urinary excretion (Brabin 1992). This would decrease iron stores and potentially induce iron deficiency. The effect of the acute phase response to infection may overlap malaria-specific changes in iron metabolism (Baynes et al. 1986). During illness, serum iron concentrations fall as iron is apparently redistributed to storage sites (Elin et al. 1977). As a consequence, hemoglobin falls and EP rises, mimicking the anemia caused by iron deficiency, but ferritin is elevated considerably. In general, on the basis of expected changes in iron status measures, hemoglobin measures would overestimate and ferritin measures would underestimate the prevalence of iron deficiency in a malaria-endemic population.

To determine whether malarial infection influenced indicators of iron deficiency in asymptomatic children, we examined relationships among hemoglobin, EP, ferritin and malaria parasitemia in 3605 children attending schools on Pemba Island, Zanzibar, Tanzania. Coastal East Africa, including the islands of Zanzibar, has the highest malaria endemicity in the world. Plasmodium falciparum malaria is holoendemic, that is, parasitemia is highly prevalent but of low density, and transmission is relatively stable, with little seasonality of prevalence of infection (Marsh 1992). This article will specifically address the following questions regarding the relationships of iron status indicators and malaria parasite density in these children: 1) Is hemoglobin, EP or ferritin or the prevalence of iron-deficiency anemia associated with the level of malaria parasitemia? 2) Does malaria parasitemia obscure the expected relationships among these indicators of iron status? 3) Does malarial infection bias estimates of the population prevalence of iron deficiency or iron deficiency anemia using these indicators?

MATERIALS AND METHODS

Table 1. Expected relationships among iron status indicators during iron deficiency and as a consequence of malaria pathology [View Table]

Table 2. Characteristics of study sample [View Table]

The relationships of hemoglobin, EP and ferritin to malaria parasitemia level are expressed using indicator means and selected cutoffs to define deficiency. Distributions of EP and ferritin were skewed to high values; therefore geometric means are presented. Anemia was defined as hemoglobin <110 g/L. This anemia cutoff is lower than the WHO-recommended cutoff for this age group, but a race-specific anemia criterion (10 g/L lower for blacks) optimizes the screening performance of this indicator to detect iron deficiency (Johnson-Spear and Yip 1994). Ferritin values <12 µg/L were considered to indicate exhausted iron stores, and an EP value of >80 µmol/mol heme was used to indicate iron-deficient erythropoiesis (WHO/UNICEF/UNU 1995). Iron-deficiency anemia was defined as abnormal values for hemoglobin, EP and serum ferritin. Linear trends in mean values were tested by linear regression, and trends in proportions were tested by chi-square test for trend (Snedecor and Cochran 1980).

Table 3. Means and prevalence of abnormal values of iron status indicators by level of malaria parasitemia in children [View Table]

To evaluate whether the relationships among ferritin, EP and hemoglobin levels differed by low and high parasitemia, we stratified the sample using a cutoff for parasite density of 1000 parasites/µL. Various cutoffs for level of parasitemia were considered, and this cutoff was chosen because it maximized the fit of the model when ferritin or EP was regressed on hemoglobin, level of parasitemia, and their interaction term. Means, SD, and proportions of abnormal values were examined for EP by level of hemoglobin, and ferritin by level of hemoglobin or EP. To determine whether linear trends in these indicators by category were statistically significant for both high and low malaria groups, the indicator was regressed on hemoglobin or EP, malaria level, and their interaction term. The significance level of the interaction term was used to determine whether the relationship between indicators differed by malaria level, with P < 0.15 indicating a potentially important difference.

To evaluate the bias caused by malarial infection in evaluating iron status of this population, we compared mean values and the percentage of abnormal values of each indicator in the total sample to those of the children with lower malaria parasitemias. These were tested by Student's t test and chi-square test, respectively (Snedecor and Cochran 1980).

Data were entered using EpiInfo software (Centers for Disease Control and Prevention, Atlanta, GA) and managed and analyzed using Systat statistical software (SYSTAT Inc., Evanston, IL).

RESULTS

Association between malaria parasitemia and hemoglobin erythrocyte protoporphyrin and ferritin concentrations. Hemoglobin and EP concentrations were not associated with malaria parasitemia (Table 3). The proportion of abnormal values for both hemoglobin and EP showed significant increasing trends with ascending level of malaria parasitemia. Both trends were similar in magnitude, with the prevalence of abnormal values in infected children reaching a maximum of 125% of the prevalence of children with no parasitemia. The highest prevalence of abnormal values was, however, not found in the highest malaria parasitemia group.

Ferritin concentration demonstrated no relationship with parasitemia at parasite densities <1000 parasites/µL blood, but above this cutoff ferritin increased slightly at a rate of 1.5 µg/L per 1000 parasites. This cutoff was used to define high and low malaria parasitemia in subsequent analyses. The prevalence of abnormal ferritin values began to fall at malaria parasite densities >2000 parasites/µL. Both the trend in ferritin concentration and prevalence of abnormal values were significant. The prevalence of iron-deficiency anemia did not show a significant linear trend with malaria parasite density but was dramatically lower in the 30 children with 4000 parasites/µL.

Table 4. The influence of high malaria parasitemia on the relationship of erythrocyte protoporphyrin to hemoglobin (Hb) levels in children [View Table]

Ferritin was shifted significantly toward higher values when malaria parasitemia was >1000 parasites/µL; this shift is reflected in increases in mean values and decreases in the percentage of children with ferritin <12 µg/L. Nonetheless, the positive association between ferritin and hemoglobin and the negative association between ferritin and EP were strong regardless of malarial infection (Table 5).

Table 5. The influence of high malaria parasitemia on the relationship of serum ferritin to hemoglobin (Hb) and erythrocyte protoporphyrin (EP) levels in children [View Table]

These analyses were repeated using malaria parasite density cutoffs of 1 parasite/µL (i.e., any parasitemia) and 2000 parasites/µL (data not shown). The relationships between the indicators was not modified by malaria using any parasite density cutoff.

DISCUSSION

We cannot evaluate the influence of malarial infection on other indicators, such as transferrin saturation or serum transferrin receptor, because they were not assessed in this survey. A similar evaluation of these indicators in a malaria-endemic population is needed. The three indicators used in this survey were chosen because of their simplicity in the field (in the case of hemoglobin and EP), long-term stability at 20°C (in the case of serum ferritin), relatively low cost to determine, and their complementarity across the spectrum of iron status. Because of these advantages, it is encouraging to find that these indicators are useful in this population.

Few data are available on the effect of malaria on EP, and we found no published data on EP concentrations during acute malarial disease. In theory, EP values could be elevated in response to infection, as observed in chronic tuberculosis (Langer et al. 1972) and chronic inflammatory neoplastic diseases (Hastka et al. 1993), or decreased due to the dyserythropoiesis that occurs during malaria. In this study, the relationship of EP to hemoglobin was somewhat attenuated in children with higher parasite densities. In contrast to our findings, Schneider et al. (1993) found that mean EP concentration was elevated in children in Togo aged 6 mo to 3 y with parasitemia >3000 parasites/µL. Prevalence of anemia was not associated with parasitemia in those children. The authors noted that the hemolytic breakdown products of heme fluoresce similarly to EP, and speculate that the elevation in EP associated with malaria is artifactual rather than caused by true changes in erythropoiesis. Although the discrepancy of our findings with those of Schneider et al. (1993) is unresolved, the effect of parasite density on EP was not an important consideration when evaluating iron status at the population level in these children.

The most important finding was that the relationship of ferritin to hemoglobin and EP was not affected by malaria parasitemia, even though the magnitude of ferritin values was greater when parasitemia was >1000 parasites/µL. Even in individuals in our population with high parasite densities, ferritin was directly related to hemoglobin and inversely related to EP. Had malaria been a major contributor to anemia, we would have expected the normal relationship between ferritin and degree of anemia to be obscured (Phillips et al. 1986) or reversed (Adelekan and Thurnham 1990), because sicker individuals typically have lower hemoglobin and higher ferritin. Although ferritin values increased slightly with parasite densities >1000 parasites/µL, ferritin was still well below values reported for individuals with acute malaria. For example, the mean ferritin concentration at admission among 23 individuals hospitalized with acute, symptomatic, uncomplicated P. falciparum malaria was 1773 µg/L (range 170-10,000 µg/L). Ninety days after initiation of treatment, all values had returned to 150 µg/L or below (Phillips et al. 1986). Only five individuals in our population had ferritin >200 µg/L. At the population level, endemic malarial infection did not affect the prevalence of abnormal ferritin values.

Our findings are in contrast to studies that examined the relationships of malaria infection and iron status indicators in two types of populations: those acutely ill with malaria, in whom iron status indicators are dramatically perturbed (see above), and in young children. Bradley-Moore et al. (1985), in a study of Nigerian children aged 3 mo to 2 y, found that children with parasitemia had around 10 g/L lower hemoglobin and approximately doubled ferritin concentrations compared with their peers without parasitemia. The prevalence of parasitemia in this community was 41%. An age-related effect is also supported by studies in Papua New Guinea (Spencer 1966) and The Gambia (McGregor et al. 1966), in which the association of malaria parasitemia with hemoglobin concentration disappeared with increasing age. Among infants in both studies, children with parasitemia had hemoglobin concentrations 10-20 g/L lower than did their peers without parasitemia. Among children aged 2-4 y, parasitemia was associated with 12 g/L lower hemoglobin concentration in Papua New Guinea but was not associated with lower hemoglobin concentration in The Gambia. Among Papua New Guinean children aged 5-9 y, the association of parasitemia with hemoglobin concentration was slight (a reduction of 3 g/L). Finally, Premji et al. (1995) also found a strong association between anemia (measured by hematocrit) and parasitemia, which decreased with age in 6- to 40-mo-old children in coastal Tanzania, an area expected to have transmission patterns of the parasite similar to those in Zanzibar. Further studies are needed to document, however, whether the effect of parasitemia on other iron status indicators shows a similar disappearance with age in holoendemic environments.

Different patterns of malaria transmission are broadly categorized as holoendemic, hyperendemic and epidemic, according to intensity and stability of transmission (Najera et al. 1993). The immunity of the population, and thus the probability that infection is associated with disease, differs greatly depending on these patterns. In holoendemic settings such as Zanzibar, parasitemia is prevalent and of high density and splenomegaly is common in infants and young children; however, in adults, malaria parasitemia is prevalent but of low density. Because of the immunity to infection that is acquired in holoendemic settings, the distribution of malarial infection may differ dramatically from that of malarial disease across age groups and seasons (Marsh 1992).

Finally, within a given ecological context, population subgroups have different levels of immunity to the parasite. Immunity, as well as age, is dependent on factors such as general nutritional status and physiologic state, notably pregnancy (Marsh 1992).

We hypothesize that hemoglobin, EP and ferritin are valid indicators of iron status in population groups with relatively high immunity to malaria, but will be less reliable in groups who are less immune. Put in another way, iron status indicators will measure iron status independently of malaria parasitemia in population groups where parasitemia is not strongly associated with disease, which is generally the case among older children and nonpregnant adults in holoendemic environments. If true, this would be important and encouraging news for the assessment of iron deficiency in tropical Africa, where iron deficiency is a prevalent problem among school-age children and adult women. In surveys of iron status in these population groups, it will still be desirable to exclude people who are experiencing fever and those with parasite densities greater than some threshold; Trape et al. (1985) suggest a threshold of 5000 parasites/µL blood. Our data indicate that 5000 parasites/µL would be an appropriate cutoff for hemoglobin and EP. For ferritin, 1000 parasites/µL might be more appropriate, but ferritin would not bias population estimates if the proportion of individuals above such a threshold is relatively small. Further research is required to extend our findings to other vulnerable population groups.