Elevations in serum anti-flagellin and anti-LPS Igs are related to growth faltering in young Tanzanian children

Background: Antibodies to LPS and flagellin have been described as indirect measures of increased gastrointestinal permeability and may be markers of environmental enteric dysfunction (EED), which is a condition associated with poor child growth. Objective: We assessed whether LPS- and flagellin-specific immunoglobulin (Ig) concentrations were associated with poor growth in young Tanzanian children at risk of EED. Design: Blood samples were obtained from 590 children at ± wk, ± mo, and 12 mo of age. Serum LPS- and flagellin-specific Ig concentrations (IgA and IgG) were measured with the use of an ELISA. Growth was measured on a monthly basis for 18 mo. Results: Anti-LPS and anti-flagellin IgA and IgG concentrations increased over the first year of life and were higher than concentrations (measured at 9 mo of age) in healthy controls. Children with anti-flagellin IgA, anti-LPS IgA, anti-flagellin IgG, and anti-LPS IgG concentrations in the highest quartile at ± wk of age were 2.02 (95% CI: 1.11, 3.67), 1.84 (95% CI: 1.03, 3.27), 1.94 (95% CI: 1.04, 3.62), and 2.31 (95% CI: 1.25, 4.27) times, respectively, more likely to become underweight (weight-for-age z score <-22) after adjustment for covariates (P-trend < 0.05) than were children with Ig concentrations in the lowest quartile. Children with increased concentrations of anti-flagellin IgA were also more likely to become wasted; however, there was no association between any of the markers and subsequent stunting. Conclusion: Serologic measures of increased intestinal permeability to bacterial components are associated with subsequent poor growth and could help identify children who may benefit most from preventive interventions. Subjects included in this analysis were part of a randomized, double-blind, placebo-controlled trial that was designed to investigate whether the daily administration of zinc or multivitamins to Tanzanian infants reduced risk of infectious morbidity compared with that shown with the administration of a placebo. Results of this trial have been published previously (9). In brief, HIV-negative mothers of potentially eligible infants were recruited into the study, and their infants were randomly assigned to one of 4 study arms between 5 and 7 wk of age. Infants of multiple births and those with congenital anomalies or other conditions that would interfere with the study procedures were excluded from the trial. Birth characteristics were obtained immediately after delivery. At the time of random assignment, a study physician performed a clinical examination, and a study nurse performed a history of morbidity and infant-feeding practices, anthropometric measurements, and a blood draw. Mothers were asked to return to the study clinic with their infants every 4 wk for data collection and standard clinical care including growth monitoring, immunizations, routine medical treatment of illnesses, and periodic vitamin A supplementation (100,000 IU at 9 mo of age and 200,000 IU at 15 mo of age). At these visits, study nurses performed a morbidity history, measured the child’s weight with the use of a digital infant balance with a 10-g precision (Tanita), and measured the child’s length with a 1-mm precision with the use of a rigid length board with a movable foot piece. Blood samples were obtained from children at baseline and at 6, 12, and 18 mo of age. To be eligible for inclusion in this subanalysis of anti-flagellin and anti-LPS Igs and child growth, children were required to have a blood sample available at 6 wk and 6 mo of age and have a length-for-age z score (LAZ) of ≥−2 at 6 wk of age. We anticipated that 590 children would meet both eligibility criteria, which would have provided 99% power to detect a stunting HR of 1.5 for every 0.4-U increase in anti-LPS IgG and 91% power to detect a stunting HR of 1.5 for every 0.2-U increase in anti-flagellin IgG. These calculations assumed SDs of 0.48 and 0.19 for anti-LPS IgG and anti-flagellin IgG (10), respectively, an estimated stunting incidence of 30% by 2 y of age (11), and a 15% rate of loss to follow-up. Flagellin- and LPS-specific IgA and IgG concentrations were measured with the use of an ELISA as previously reported (7). Microtiter plates were coated with purified Escherichia coli flagellin (100 ng/well) or purified E. coli LPS (2 μg/well). Serum samples from study participants were diluted 1:200 and applied to wells coated with flagellin or LPS. After incubation and washing, the wells were incubated with anti-human IgA (KPL) or IgG (GE Healthcare) coupled to a horseradish peroxidase. The quantification of total Igs was performed with the use of the colorimetric peroxidase substrate tetramethylbenzidine, and absorbance (optical density) was read at 450 nm with the use of an ELISA plate reader. Data are reported as optical density–corrected data by subtracting background concentrations, which were determined from the readings in samples that lacked serum. For purposes of comparison, anti-flagellin and anti-LPS Igs were also measured in a cohort of 36 healthy infants seen at Boston Children’s Hospital. After institutional review board approval was obtained, any infant from birth to 12 mo of age with an excess blood sample in the Boston Children’s Hospital central laboratory was eligible for inclusion. Subjects were excluded from participation in the research study if 1) their LAZ was <−2 or >2; 2) their WAZ was <−2 or >2; 3) no growth data were available for the review and analysis; 4) there was evidence of a recent fever, which was defined as any temperature >38.5°C in the past 48 h; and 5) there was any evidence of a systemic illness (e.g., malignancy). A total of 67 infants were screened for enrollment, and 31 infants were excluded. The mean age of the remaining 36 infants at the time of the blood draw was 9.5 mo (range: 5–12 mo). Data were double entered with the use of Microsoft Access software (version 12.0; Microsoft Corp.), converted to SAS (version 9.2; SAS Institute) data sets, and uploaded to a secured UNIX-based server (Oracle Solaris 10 x64) for analysis. Descriptive statistics were used to summarize baseline characteristics of the study population. Frequencies were reported for categorical variables and means ± SDs for continuous variables. The possibility of nonlinear growth in children’s LAZ and weight-for-age z score (WAZ) over the course of follow-up was examined nonparametrically with restricted cubic splines (12). An automated stepwise selection with entry and retain criteria of P < 0.05 was used for the placement of 21 knots in each curve. The concentrations of each of the 4 biomarkers (i.e., anti-flagellin IgA, anti-flagellin IgG, anti-LPS IgA, and anti-LPS IgG) at 6 wk and 6 mo of age were categorized into quartiles to examine their associations with child growth. Separate Cox proportional hazards models were constructed to estimate HRs and corresponding 95% CIs with the time to the first episode of stunting, wasting, and underweight as the outcome and the child’s age (in mo) as the metameter. Each outcome was modeled separately, and the first time the child reached a score of <−2 SDs was classified as an event. The follow-up time was censored at the time of the event or the time of the last anthropometric assessment. To assess the association between each biomarker and the anthropometric outcomes, we first ran a series of univariate models. Second, we ran multivariate models that adjusted for the following covariates: child sex, preterm birth, maternal age, maternal education, maternal midupper arm circumference, number of household assets, study regimen, and anthropometric z score at 6 wk of age. All analyses were performed with the use of SAS version 9.2 software (SAS Institute). Institutional approval was granted by the Harvard T.H. Chan School of Public Health Human Subjects Committee; the Muhimbili University of Health and Allied Sciences Research and Publications Committee; the National Institute of Medical Research, Tanzania; the Tanzanian Food and Drugs Authority; and the Boston Children's Hospital Committee on Clinical Investigation. This trial was registered at clinicaltrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT00421668","term_id":"NCT00421668"}}NCT00421668.