The association of malaria morbidity with linear growth, hemoglobin, iron status, and development in young Malawian children: a prospective cohort study

The iLiNS-DOSE and iLiNS-DYAD-M studies were conducted in one public district hospital (Mangochi), one mission hospital (St Martins), and two rural public health centers (Lungwena and Namwera) in Mangochi District, Southern Malawi. The total catchment population of 180,000 largely subsisted on farming and fishing. In Malawian children aged < 5 years, the prevalence of reported fever (a proxy for malaria), diarrhea and ARI was 29, 22 and 5%, respectively, with seasonal fluctuations [17]. Malaria is endemic in Malawi and the study area has high malaria transmission with high temperature and frequent rainfall from October through April [18].

In the iLiNS-DOSE study, 6-mo old children were randomly allocated to one of five intervention groups provided with different doses or formulations of LNS or to a control group that did not receive LNS during the 12-mo study period, between November 2009 and May 2012. In the iLiNS-DYAD-M study, pregnant women < 20 weeks’ gestation were randomly allocated to one of three groups to receive iron and folic acid (IFA), multiple micronutrients (MMN) or a small-quantity (20 g) of LNS daily. After delivery, women in the IFA group received placebo tablets, while MMN and LNS supplementation was continued up to 6 mo postpartum. Children of mothers in the LNS group also received LNS 10 g twice daily from age 6 to 18 mo. This study was conducted from February 2011 to April 2015. Details of study design, randomization and enrolment for the two studies were explained in the main outcome papers [15, 16].

In both studies, research assistants visited the children’s homes every week from age 6 to 18 mo to interview the guardians about the child’s health in the previous 7 days using a structured questionnaire. The information was complemented by a picture calendar filled out by the guardians daily to aid memory of their children’s morbidity status. The use of maternal interviews as a means of collecting data on child morbidity has been validated in previous studies [19, 20]. The research assistants referred all cases of ‘presumed’ malaria (presence of fever) to the nearby health facility for treatment with lumefantrine/artemether, the nationally recommended antimalarial drug. The children were followed throughout the year, covering periods of both high and low malaria transmission.

Anthropometric measurements were taken at age 6 mo and 18 mo. Study anthropometrists measured the infant’s length with a high-quality length board (Harpenden Infantometer; Holtain Limited) and recorded it to the nearest 1 mm. They weighed unclothed infants with electronic infant weighing scale (SECA 735; Seca GmbH & Co), recording to the nearest 10 g. The anthropometrists were trained and their measurement reliability was verified at the start of the study and at 6-mo intervals thereafter with methods adapted from the procedures used in the WHO Multicentre Growth Reference Study [21]. The anthropometrists calibrated all equipment with standard weights and length rods daily.

We assessed iron status at age 6 mo and 18 mo by measuring the zinc protoporphyrin (ZPP) concentration in unwashed venous blood sample using a hematofluorometer (206D, AVIV Biomedical Inc., Lakewood, NJ, USA). About 5–7 ml of blood was collected by venepuncture using a 23-gauge needle into 7.5 ml evacuated, trace element-free polyethylene tubes containing lithium heparin (Sarstedt AG & Co, Nümbrecht, Germany). The samples were kept covered in aluminium and away from light, in a refrigerator or on ice, and processed within 2 h of collection. We measured blood hemoglobin (Hb) concentration at age 6 mo and 18 mo from a drop of blood taken from a finger prick and collected in a microcuvette. Hb analysis was conducted on-site using a Hemo-Cue instrument (Hemocue 201+, HemoCue® AB, Ängelholm, Sweden).

We assessed fine and gross motor development at age 18 mo using the Kilifi Developmental Inventory (KDI) developed in Kenya [22]. Language development was assessed using a 100-word vocabulary checklist by maternal interview based on the MacArthur-Bates Communicative Development Inventory [23] adapted for the local languages, and 18-mo socio-emotional development was assessed using the Profile of Social and Emotional Development (PSED), also developed in Kenya. The child’s mood during the KDI assessment was rated as positive (smiling/laughing) or not positive (crying/inconsolable, changeable/mood swings, or no visible emotions). The child’s interaction with the assessor during the KDI was rated as positive (friendly) or not positive (avoidant and withdrawn, clings to family member, hesitant/when approached will accept reluctantly, difficult to engage in tasks, or inappropriate approaches to the assessor). The child’s activity level during the KDI was rated as positive (active and maintains interest) or not positive (unarousable, sleepy and can hardly be awakened, sleepy but easily awakened, does not spontaneously engage in activity, and awake but loses interest). The KDI, vocabulary, and PSED scores showed high inter-rater agreement and moderate to high test-retest reliability in this study setting [24, 25].

We used a presumptive diagnosis of malaria derived from episodes of fever during the previous week, reported by the guardians. To ensure the diagnoses were mutually exclusive, we created an algorithm whereby any fever with a diarrhea episode (three or more loose stools in 24 h) was categorized as diarrhea; any fever in the presence of any respiratory symptoms (cough, rapid or difficult breathing and nasal discharge) was categorized as ARI. ‘Presumed’ malaria was defined as any fever episode in the absence of diarrhea and respiratory symptoms.

An episode of ‘presumed’ malaria, ARI or diarrhea was defined as the period starting from the day the child had the symptoms when preceded by at least 2 days of no symptoms or no data. The episode ended on the last day the child had the symptoms which was then followed by at least 2 symptom-free days. Incidence of ‘presumed’ malaria, ARI or diarrhea for each child from age 6 to 18 mo was calculated as total episodes / total follow up years at risk.

Longitudinal prevalences of common childhood symptoms (fever, diarrhea, and cough) from age 6 to 18 mo were defined as the number of days with the symptom divided by the total number of days of observation for each child [26].

We calculated age- and sex-standardized anthropometric indices [length-for-age z score (LAZ), weight-for-age z score (WAZ), and weight-for-length z score (WLZ)] based on the WHO Child Growth Standards [21] and considered values below – 2.0 indicative of underweight, stunting and wasting, respectively. Change in LAZ for each child was calculated as the difference between LAZ at age 18 mo and LAZ at age 6 mo.

Iron deficiency at age 6 mo and 18 mo was defined as whole blood ZPP > 70 μmol/mole heme [27]. Anemia at age 6 mo was defined as blood Hb concentration < 105 g/L [28] while anemia at age 18 mo was defined as blood Hb concentration < 110 g/L [29].

From the child development data at age 18 mo, fine motor scores were calculated as the sum of 34 KDI fine motor items, each scored 0 or 1, gross motor scores were calculated as the sum of 35 KDI gross motor items, each scored 0 or 1 [22] and vocabulary score was the maternal-reported child expressive vocabulary out of the 100-word checklist. For these outcomes, moderate to severe delay was defined as the bottom 25% of the sample. The socio-emotional score was calculated as the sum of 19 PSED items. Moderate to severe delay was defined as the top 25% of our sample (a higher score indicates less advanced socio-emotional development).

We included in the analysis children who had outcomes measured at age 18 mo. For all continuous outcomes, we used ordinary least squares regression to assess the association between malaria incidence and each outcome; and for all binary outcomes, we used modified Poisson regression (with a robust variance estimator) [30].

We first assessed whether the relationship between the predictor and each outcome differed between the two studies. However, the interaction term was not statistically significant indicating that this relationship was not different between the two studies therefore we pooled data from the two cohorts. We then constructed multivariate models to determine which variables independently predicted the outcomes. We included all theoretically relevant variables, regardless of whether they were statistically significant or not after the bivariate analysis. The following variables collected at age 6 mo were included in the models: child sex, LAZ, WLZ, Hb, iron status, maternal education and household food insecurity access (HFIA) score generated by summing the value of responses to nine questions regarding food insecurity [31]. We also included in the models, from age 6 to 18 mo, the incidence of diarrhea and ARI, and whether the child received an intervention (LNS) or not. For the risk of stunting at age 18 mo, we included in the model stunting at age 6 mo (in place of LAZ). In addition, all developmental outcomes were adjusted for the child’s mood, activity level, age and interaction with the assessor.

We assessed collinearity among the variables (e.g. LAZ vs WLZ at age 6 mo). If the variables were highly collinear (> 0.5), we dropped the one that was less strongly associated with the outcomes. We accounted for intracluster correlation due to twins using generalised estimating equations [32].

We also performed exploratory analyses by using frequency of malaria episodes (from age 6 to 18 mo) as a categorical variable (no episode, one episode, and > 1 episodes groups). In addition, we conducted stratified analyses by stunting at age 6 mo. Although we performed bivariate analyses for each individual variable, we will only report the results from multivariate analysis.

We used Stata version 14 (StataCorp, Texas, USA) for all the analyses.

Of the 2723 children enrolled in the two study cohorts, 2016 (74.0%) had length measured both at age 6 mo and 18 mo (1417 children from the iLiNS DOSE study and 599 children from the iLiNS DYAD-M study). These were included in the final analysis (Fig. 1). The characteristics of these children at age 6 mo are summarized in Table 1.

The 2016 children included in the final analysis contributed 1647.9 child years of follow up, i.e. the mean (standard deviation) [SD] duration of follow up was 298 (61) days / child. A total of 24,024 morbidity episodes were reported during the home visits. Of these, 9.7% (2324/24024) were episodes of ‘presumed’ malaria. The rest of the morbidity episodes were due to: acute respiratory infections (ARI), 55.6% (13,360/24024); diarrhea, 33.6% (8083/24024); and minor conditions, 1.1% (257/24024).

Overall, the mean (SD) incidence of all illnesses combined was 14.8 (6.8) episodes per child year. The mean (SD) incidence of ‘presumed’ malaria was 1.4 (2.0) episodes per child year. The mean (SD) incidence of ARI was 8.3 (5.0) episodes per child year and the mean (SD) incidence of diarrhea was 4.6 (10.1) episodes per child year. The longitudinal prevalences of common childhood symptoms (fever, diarrhea, and cough) from age 6 to 18 mo were: 7.5%; 3.4%; and 11.7%, respectively.

During the 12-mo follow up period, 39.0% (787/2016) of the children did not report any episode of ‘presumed’ malaria, 30.3% (611/2016) reported one episode and 30.7% (618/2016) reported > 1 malaria episodes. The children who reported > 1 malaria episodes were responsible for 73.7% (1713/2324) of all ‘presumed’ malaria episodes reported in the two studies.

At age 18 mo, the mean (SD) length-for-age z-scores (LAZ), weight-for-age z-scores (WAZ) and weight-for-length (WLZ) scores were − 1.8 (1.1), − 1.0 (1.1) and − 0.2 (1.1) respectively. The proportions of children who were stunted, underweight and wasted were 41.5, 16.6 and 5.0%, respectively. The median (25th, 75th centile) zinc protoporphyrin (ZPP) concentration was 74 (51, 114) μmole/mole heme and the proportion with iron deficiency was 54.1%. The mean (SD) hemogobin (Hb) concentration was 108.5 (15.1) g/L and the proportion with anemia was 50.5%. The mean (SD) scores for fine motor, gross motor, language and Profile of Social and Emotional Development (PSED) were 20.9 (2.2), 17.3 (2.6), 26.2 (5.0) and 16.2 (5.4) respectively.

The mean (SD) change in LAZ from age 6 to 18 mo was − 0.44 (0.77). In multivariate analysis, there was no association between the incidence of ‘presumed’ malaria and change in LAZ from age 6 to 18 mo, adjusted for LAZ at age 6 mo (B = − 0.02, 95% CI = − 0.04 to 0.01, p = 0.069) (Table 2).

The proportion of children who were stunted increased from 27.4% at age 6 mo to 41.5% at age 18 mo. In multivariate analysis, the incidence of ‘presumed’ malaria was associated with higher risk of stunting at age 18 mo, adjusted for stunting at age 6 mo (RR = 1.04, 95% CI = 1.01 to 1.07, p = 0.023) (Table 2). When categorized by frequency of malaria episodes and adjusted for stunting at age 6 mo, children with > 1 malaria episodes from age 6 to 18 mo had higher risk of stunting at age 18 mo compared to children with zero malaria episodes (RR = 1.39, 95% CI = 1.13 to 1.70, p = 0.002).

The incidence of ‘presumed’ malaria from age 6 to 18 mo was associated with higher risk of anemia at age 18 mo, adjusted for hemoglobin at age 6 mo (RR = − 0.12; 95% CI = − 0.20 to − 0.04; p = 0.002) but not with hemoglobin or iron deficiency at age 18 mo (Table 3).

The association of incidence of ‘presumed’ malaria from age 6 to 18 mo with child development was significant for PSED scores (B = − 0.21; 95% CI = 0.39 to − 0.03; p = 0.041), but not for the other domains of child development, adjusted for the covariates listed in the footnotes to Tables 4 and 5.

In multivariate analysis, incidence of diarrhea from age 6 to 18 mo was associated with change in LAZ from age 6 to 18 mo (B = − 0.02; 95% CI = − 0.03 to − 0.01; p = 0.009), higher risk of stunting at age 18 mo (RR = 1.02; 95% CI = 1.01 to 1.03; p = 0.005) (Table 2), lower gross motor scores at age 18 mo (B = − 0.02; 95% CI = − 0.03 to − 0.01; p < 0.001), and higher risk of gross motor delay (RR = 1.01; 95% CI = 1.00 to 1.02; p < 0.001) and fine motor delay (RR = 1.01; 95% CI = 1.00 to 1.02; p = 0.011) at age 18 mo (Tables 4 and 5).

The incidence of ARI from age 6 to 18 mo was not significantly associated with growth or other outcomes except for PSED scores (B = 0.08; 95% CI = 0.03 to 0.14; p = 0.004), and PSED delay (RR = 1.02; 95% CI = 1.00 to 1.04; p = 0.025) (Tables 4 and 5).