Putative Biomarkers of Environmental Enteric Disease Fail to Correlate in a Cross-Sectional Study in Two Study Sites in Sub-Saharan Africa

Abstract

Environmental enteric dysfunction (EED) is an elusive, inflammatory syndrome of the small intestine thought to be associated with enterocyte loss and gut leakiness and lead to stunted child growth. To date, the gold standard for diagnosis is small intestine biopsy followed by histology. Several putative biomarkers for EED have been proposed and are widely used in the field. Here, we assessed in a cross-sectional study of children aged 2-5 years for a large set of biomarkers including markers of protein exudation (duodenal and fecal alpha-1-antitrypsin (AAT)), inflammation (duodenal and fecal calprotectin, duodenal, fecal and blood immunoglobulins, blood cytokines, C-reactive protein (CRP)), gut permeability (endocab, lactulose-mannitol ratio), enterocyte mass (citrulline) and general nutritional status (branched-chain amino acids (BCAA), insulin-like growth factor) in a group of 804 children in two Sub-Saharan countries. We correlated these markers with each other and with anemia in stunted and non-stunted children. AAT and calprotectin, CRP and citrulline and citrulline and BCAA correlated with each other. Furthermore, BCAA, citrulline, ferritin, fecal calprotectin and CRP levels were correlated with hemoglobin levels. Our results show that while several of the biomarkers are associated with anemia, there is little correlation between the different biomarkers. Better biomarkers and a better definition of EED are thus urgently needed.

Keywords: Sub-Saharan Africa; alpha-1-antitrypsin; anemia; biomarker; calprotectin; citrulline; environmental enteric dysfunction; insulin-like growth factor; lactulose-mannitol test; stunted child growth.

Figure 1

Subjects and samples included in this study: (A) flowchart of the subjects included in this study; (B) summary of the samples available for each biomarker/ combination of biomarkers.

Figure 2

Correlation of different biomarkers between body compartments, with each other as well as with the number of virulence genes: (A) correlation plot of calprotectin levels in feces and duodenum; (B) correlation plot of alpha-1-antitrypsin (AAT) levels in feces and duodenum; (C) number of enteropathogenic virulence genes detected according to calprotectin and AAT levels; (D) calprotectin and AAT concentration measured in the feces depending on the presence of at least one virulence gene measured by qPCR; (E) correlation of levels of fecal calprotectin and AAT levels; (F) correlation of citrulline levels and the geometric mean of branched-chain amino acids (BCAA = isoleucine, leucine, valine); (G) correlation of blood citrulline and CRP levels; correlations are based on non-parametric Spearman correlation; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 3

Factors associated with putative biomarkers of EED. Regression models are shown per biomarker, considering associations with other biomarkers as well as potential confounding factors. Significant associations (p < 0.05) are indicated in blue.

Figure 4

Putative biomarkers and their association with growth delay: (A) association of different biomarkers with height-for-age z-score; (B) blood amino acid levels as a function of stunting status; (C) regression results of the main biomarkers and potential confounders and stunting; (D) regression results of the main biomarkers and potential confounders including branched-chain amino acids (BCAA) and stunting. Significant associations (p < 0.05) are indicated in blue. Correlations are based on non-parametric Spearman correlation; ***: p < 0.01, ns: p > 0.05.

Figure 5

Correlation between putative EED biomarkers and hemoglobin levels.