Biomarkers of systemic inflammation and growth in early infancy are associated with stunting in young Tanzanian children

Stunting can afflict up to one-third of children in resource-constrained countries. We hypothesized that low-grade systemic inflammation (defined as elevations in serum C-reactive protein or alpha-1-acid glycoprotein) in infancy suppresses the growth hormone–insulin-like growth factor (IGF) axis and is associated with subsequent stunting. Blood samples of 590 children from periurban Dar es Salaam, Tanzania, were obtained at 6 weeks and 6 months of age as part of a randomized controlled trial. Primary outcomes were stunting, underweight, and wasting (defined as length-for-age, weight-for-age and weight-for-length z-scores < −2) between randomization and endline (18 months after randomization). Cox proportional hazards models were constructed to estimate hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of time to first stunting, underweight, and wasting as outcomes, with measures of systemic inflammation, insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) as exposures, adjusting for numerous demographic and clinical variables. The incidences of subsequent stunting, underweight, and wasting were 26%, 20%, and 18%, respectively. In multivariate analyses, systemic inflammation at 6 weeks of age was significantly associated with stunting (HR: 2.14, 95% CI: 1.23, 3.72; p = 0.002). Children with higher levels of IGF-1 at 6 weeks were less likely to become stunted (HR: 0.58, 95% CI: 0.37, 0.93; p for trend = 0.019); a similar trend was noted in children with higher levels of IGF-1 at 6 months of age (HR: 0.50, 95% CI: 0.22, 1.12; p for trend = 0.07). Systemic inflammation occurs as early as 6 weeks of age and is associated with the risk of future stunting among Tanzanian children. Subjects were part of a randomized, double-blind, placebo-controlled trial designed to investigate whether daily administration of zinc and/or multivitamins (vitamin C, vitamin E, thiamin, riboflavin, niacin, vitamin B6, folate, and vitamin B12) to Tanzanian children would reduce the risk of infectious morbidity and improve growth compared with placebo. Results of this trial have been published previously [17]. Briefly, HIV-uninfected mothers of potentially eligible children were recruited into the study and their infants were randomized to 1 of the 4 study arms between 5 and 7 weeks of age. Children of multiple births and those with congenital anomalies or other conditions that would interfere with the study procedures were excluded from the trial. Subjects were followed for 18 months. Birth characteristics were obtained immediately after delivery whenever possible. At the time of randomization, a study physician performed a clinical examination and drew a blood sample, and a study nurse performed a history of morbidity and infant feeding practices and anthropometric measurements. Mothers were asked to return to the study clinic with their children every 4 weeks for data collection and standard clinical care, including growth monitoring, immunizations, routine medical treatment for illnesses, and periodic vitamin A supplementation (100,000 IU at 9 months and 200,000 IU at 15 months). At these visits, study nurses performed a morbidity history based on maternal recall aided by the mother’s symptom diary that she received at the previous visit. The symptom diary was a pictorial aid of illness symptoms (e.g., diarrhea, vomiting) where mothers were asked to check off which days their child had experienced these symptoms. The study nurses also measured the children’s weight using a digital infant balance with 10 g precision (Tanita) and their length with 1 mm precision using a rigid length board with a movable foot piece. Length-for-age z-scores (LAZ), weight-for-length z-scores (WLZ), and weight-for-age z-scores (WAZ) were calculated by using World Health Organization (WHO) child growth standards [18]. Stunting, wasting, and underweight were defined as a LAZ, WLZ, and WAZ of 2 or more standard deviations (SDs) below the WHO population median, respectively. For the current post hoc analysis, a subset of children were chosen if they: (1) had blood samples available at 6 weeks and 6 months of age, and (2) were not stunted (LAZ ≥ −2) at 6 weeks of age (Figure 1). Flowchart demonstrating the selection criteria for our patient population. Blood samples were obtained from children at baseline, 6 weeks, and 6 months of age. Samples were centrifuged and plasma removed within 2 h of blood collection; aliquots were stored in –80 °C freezers until shipped on dry ice for analysis. Serum samples were tested for biomarkers of growth (IGF-1, IGFBP3) and systemic inflammation (AGP, CRP). IGF-1, IGFBP-3, CRP, and AGP were measured by an ELISA method from R&D Systems (Minneapolis, MN, USA). Data were double entered using Microsoft Access software (Microsoft Corporation, Redmond, WA, USA) and then converted to SAS datasets and uploaded to a secured Unix-based server for analysis. Descriptive statistics were used to summarize baseline characteristics of the study population. FrequDencies were reported for categorical variables and the mean ± standard deviation (SD) for continuous variables. The concentrations of IGF-1 and IGF-BP3 at 6 weeks and 6 months of age were categorized into quartiles and then used to examine their association with child growth. CRP concentrations increase quickly in response to acute insult, peaking at approximately 48 h and decreasing within a week, with a half-life of 19 h [19]. In contrast, AGP concentrations increased more slowly and remained elevated for a longer period of time [20]. Taken together, CRP and AGP measurements can be used to classify individuals who have inflammation from incubation to early convalescence and into late convalescence [21]. Therefore, we explored the association of inflammation with growth by defining systemic inflammation as a composite variable of either CRP > 5 mg/L [22] or AGP > 1 g/L [23] and also by categorizing each CRP and AGP individually as follows: CRP > 5 mg/L [22] or AGP > 1 g/L [23]. Cox proportional hazard models were constructed to estimate hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) for time to stunting, wasting, and underweight across the quartile category of each biomarker and the presence of any inflammation. p-value for trend was calculated by including the median value within each quartile as a continuous term in the regression model. Each growth outcome was modeled separately, and the first time the child reached a score of <−2 SD signified an “event.” Children who did not develop an “event” were censored from the study at the time of last anthropometric assessment. In multivariate analysis, we further adjusted for potential confounders including child sex, preterm birth, maternal age, maternal mid-upper arm circumference (MUAC), maternal education, number of household assets (from a list that included sofa, television, radio, refrigerator, and fan), treatment arm of parent trial, anthropometric z-score at baseline, diarrhea, malaria, unscheduled clinical visits, or hospitalization. These covariates were selected based on a p value < 0.10 in univariate analysis or traditionally considered as a risk factor for child growth outcomes in the literature or biological pathway plausibility. We adjusted for morbidity variables (e.g., diarrhea, malaria, unscheduled clinical visits, hospitalization) in the analysis because we were aware of the possibility that the relationship between inflammation and poor growth may be confounded by the high prevalence of infectious morbidities, which we previously reported in our cohort [24]. Missing data were retained with use of the missing indicator method [25]. p-values were 2-sided, with p < 0.05 considered statistically significant. All analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA). Institutional approval was granted by the Harvard T.H. Chan School of Public Health Human Subjects Committee (HSPH IRB 12875-129), the Muhimbili University of Health and Allied Science Committee of Senate Research and Publications, the National Institute of Medical Research of Tanzania, and the Tanzania Food and Drug Authority, and the parent trial was registered at clinicaltrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT00421668","term_id":"NCT00421668"}}NCT00421668. All mothers provided written informed consent for the parent trial and subsequent analyses.