Effect of a short course of iron polymaltose on acquisition of malarial parasitaemia in anaemic Indonesian schoolchildren: a randomized trial

Nangapanda village, Ende district, East Nusa Tenggara Province, Flores Island, Indonesia is a semi-urban village consisting of 18 sub-villages with an estimated population of 22,000 residents. It has a humid climate with an average yearly rainfall of around 1.800 mm and on average 82 rainy days. The rainy season lasts from November until April with a peak in rain during December to March. The primary general health care services are provided in a local Puskesmas (health centre). Plasmodium falciparum and Plasmodium vivax transmission in this area is stable enough to induce clinical immunity [14]. A population based survey using real-time PCR in the dry season in 2008 showed a prevalence of P. falciparum, P. vivax, and Plasmodium malariae of 14.5, 13.2, and 1.9% respectively [14]. Sub-microscopic parasitaemia was found in more than 80% of all positive cases. Intestinal helminth infections also have a high prevalence in this area [15].

This study was designed as a prospective double blind placebo-controlled randomized trial. It was conducted between July 2011 and January 2012. Children aged 5–18 years were recruited from twenty-one schools in Nangapanda area. Inclusion criteria were a negative malaria blood slide and mild anaemia, defined as a Hb level 8–11.4 g/dl in children below 7 years old, Hb level 8–11.9 g/dl for girls ≥7 years old and Hb 8–12.9 g/dl for boys ≥7 years old. These cut off levels were based on the 2011 criteria used by the Indonesian national government referral hospital Cipto Mangunkusumo. Exclusion criteria were clinical symptoms suggestive of malaria or any other underlying medical condition.

Enrolled children were treated with the anthelminthic drug albendazole (PT Indofarma Pharmaceutical, Bandung, Indonesia) 400 mg OD for three consecutive days. Subsequently, they were randomized in a 1:1 ratio to receive either iron(III)-hydroxide polymaltose complex (Maltofer^®, Combiphar, Bandung, Indonesia) or placebo, both taken 6 days a week for 8 consecutive weeks. The iron(III)-hydroxide polymaltose complex tablets of 357 mg contain 100 mg of elemental iron. A weight-based dosage was used aiming at a daily dose of elemental iron of 6 mg/kg daily. Randomization (simple random allocation) was performed using a computer-generated randomization list. The randomization list was created by researchers not involved in this trial, and the study doctors handed out the study medication using this list. Trial participants and laboratory personnel were blinded for treatment assignment; the study doctor was not blinded. The treatment phase started approximately 4 weeks after administration of albendazole and was followed by a follow up phase of another 8 weeks. Teachers handed out the study medication on a daily basis and documented compliance under supervision of the study clinician.

Children were followed for 96 days (16 weeks) and were reviewed by study clinicians at week 0, 4, 8, 12, and 16 following start of IPM or placebo. These study visits included measurement of body temperature and collection of venous blood, which was used for a full blood count, malaria slide (week 0, 4, 8, 12 and 16) and real-time PCR detection of malarial parasitaemia and serum ferritin concentrations (latter two at week 0 and 16). The primary outcome measure was the detection of microscopically positive malarial parasitaemia at one of the four time points and the number of RT-PCR positive children at week 16. Secondary outcome measurements were the change in Hb and ferritin concentration and MCV value. Malaria slides were examined after the study had ended and results had therefore no clinical consequences for the participating children. With the exception of the full blood count, the other laboratory examinations were also performed after ending of the study. Children with any sign of illness during the study were referred to the Puskesmas.

Presence of microscopic parasitaemia was examined by Giemsa staining of both thin and thick blood smears at the Department of Parasitology, University of Indonesia. An aliquot of 200 μl stored whole blood was used for Plasmodium DNA isolation, prior to real-time PCR detection using Plasmodium-specific primers and species specific probes, as previously described [16]. Haemocytometry was performed using a Sysmex haematological analyzer KX-21 N (Sysmex, Kobe, Japan). A MCV value below 75 fl represented microcytosis. The Mentzer index, which is the ratio between MCV and RBC count, was used as a screening test for thalassaemia using a cut off level <13 being suggestive for thalassaemia [17]. Iron deficiency was defined as ferritin level <30 µg/l, as recommended by the World Health Organization in malaria endemic regions [18].

The study was approved by the ethical committee from the Faculty of Medicine, University of Indonesia (Ref: 96/PT02.FK/ETIK/2010) and all procedures followed were in accordance with the Helsinki Declaration. Parents or caretakers of all children provided written informed consent.

Figure 1 shows details on the enrolment and follow-up. Data from 591 schoolchildren, of whom 294 were assigned to the IPM group and 297 to the placebo group, were analysed according to intention to treat. The mean age was 10 years with range of 5–18 years and 76% children were male. Baseline characteristics of the children assigned to the two groups were similar (Table 1), including gender, age distribution, weight and values of Hb, ferritin and MCV. Overall, the number of children with a positive PCR for malaria was low (5.8%), with a significantly higher proportion of children with a positive P. falciparum PCR in the placebo group than in the iron group (3.7 vs 1.0%; P = 0.03). Because of the low prevalence of submicroscopic parasitaemia at baseline, the PCR results for P. falciparum and P. vivax were combined (referred to as PCR Plasmodium) for further analyses. The proportion of children with absolute iron deficiency, defined as a serum ferritin level below 30 µg/l or 12 µg/l, was 26.9 and 8.6%, respectively. In contrast, microcytosis, defined as a MCV value below 75 fl, was present in over half (54.3%) of children. Using the Mentzer index (ratio MCV/RBC count below 13) as a screening test for thalassemia, this condition appeared common with an estimated overall prevalence of 23.0%.

A total of 13 children (10 from IPM group and 3 placebo group) were seen in between the regular study visits at the local health centre because of different clinical symptoms (Additional file 1). They were not reviewed in a systematic way. Fever was among the symptoms in 4 children in the IPM group and one child in the placebo group. Because a final diagnosis was not established in these children and malaria diagnostics were not performed, they were censored from the time-point they presented to the local health centre.

Table 2 shows data of Hb, ferritin and MCV at week 16 and the change in these parameters from baseline. Changes in values of Hb, serum ferritin and MCV were small and similar in both groups. Even in the subgroup of children with absolute iron deficiency at baseline, the increase in serum ferritin was small and similar between the groups. There was also no difference in the proportion of children remaining iron deficient at week 16 between the IPM and placebo groups.

Figure 2 shows that children assigned to the IPM group had a significantly higher rate of development of microscopically detectable parasitaemia than those assigned to the placebo group (unadjusted hazard ratio (HR) 2.2, 95% CI 1.2–4.0; P = 0.01). The cumulative number of children developing microscopic positive parasitaemia between week 0 and 16 was 29 (19 P. falciparum, 10 P. vivax) in the IPM group and 14 (8 P. falciparum, 6 P. vivax) in the placebo group. At the end of the study, 89% of the children in the IPM group had remained free from microscopically detectable parasitaemia vs 95% of children in the placebo group. Seven children had fever (≥38 °C) at one of the four regular study visits time; three of these children had a positive malaria slide on the same time point. Exploratory analysis showed that when children were stratified by iron status, the increased rate of microscopic malaria in the IPM group was due to the increased rate in children who were iron replete (serum ferritin ≥30 μg/l) at baseline (Fig. 2b). Iron deficient children in the IPM group had a similar incidence of microscopically detectable parasitaemia as children in the placebo group (95% of children free of parasitaemia during study). When children were stratified by MCV category, those with microcytosis (MCV value <75 fl) had the highest rate of developing microscopically detectable parasitaemia (Fig. 2c). Univariate and multivariate adjusted hazard ratio for the first episode of microscopic malaria were also calculated using Cox regression with treatment arm, baseline iron status, baseline MCV value and age as variables (Table 3). Assignment to the iron group and microcytosis were independently associated with an increased rate of microscopic malaria, while baseline iron status and age were not.

From the children with submicroscopic malaria at baseline (16 in the iron group and 18 in the placebo group), only 3 developed microscopically detectable parasitaemia during follow up (2 in the IPM group and 1 in the placebo group). At the end of the study, 5 children in the IPM group and 7 in the placebo group had remained PCR positive for Plasmodium. Results of the Plasmodium PCR at week 16 were available in 278 of children in the IPM group and 286 of children in the placebo group and are summarized in Table 4. A positive PCR result was found in 46 (16.6%; 23 P. falciparum, 18 P. vivax, 5 mixed infection) of children in the IPM group and in 41 (14.3%; 23 P. falciparum, 14 P. vivax, 4 mixed infection) in the placebo arm (P = 0.47). Analysing only iron-replete children, we found that 41 (20.0%; 20 P. falciparum, 16 P. vivax, 5 mixed infection) out of 205 children in the IPM group and 28 (13.3%; 15 P. falciparum, 10 P. vivax, 3 mixed infection) out of 210 children in the placebo group (P = 0.07) were PCR Plasmodium positive. Overall, a higher proportion of children with microcytosis at baseline had a positive Plasmodium PCR at week 16 compared with children with a normal MCV value (19.3 vs 9.3%; P = 0.001). However, in the subgroups of children with or without microcytosis, the proportion of Plasmodium PCR positive children was similar in the IPM and placebo arms.