See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/340153903

Penalized Regression Models to Select Biomarkers of Environmental

Enteropathy Associated with Linear Growth Acquisition in a Peruvian Birth
Cohort

Article in SSRN Electronic Journal · January 2019

DOI: 10.2139/ssrn.3360092

CITATIONS READS

0 30

12 authors, including:

Josh M. Colston Peter S Kosek

University of Virginia PeaceHealth
54 PUBLICATIONS 654 CITATIONS 35 PUBLICATIONS 1,364 CITATIONS

SEE PROFILE SEE PROFILE

Francesca Schiaffino Fahmina Fardus-Reid

Universidad Peruana Cayetano Heredia Imperial College London
39 PUBLICATIONS 238 CITATIONS 14 PUBLICATIONS 352 CITATIONS

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Josh M. Colston on 19 June 2022.

The user has requested enhancement of the downloaded file.

 RESEARCH ARTICLE

Penalized regression models to select

biomarkers of environmental enteric
dysfunction associated with linear growth
acquisition in a Peruvian birth cohort
Josh M. Colston ID1, Pablo Peñataro Yori2, Lawrence H. Moulton3, Maribel Paredes
Olortegui4, Peter S. Kosek5, Dixner Rengifo Trigoso4, Mery Siguas Salas4,
Francesca Schiaffino3, Ruthly François3, Fahmina Fardus-Reid6, Jonathan R. Swann6,
Margaret N. Kosek ID2*
a1111111111
1 Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore,
a1111111111
Maryland, United States of America, 2 Division of Infectious Diseases and International Health, University of
a1111111111 Virginia School of Medicine, Charlottesville, Virginia, United States of America, 3 Department of International
a1111111111 Health, Johns Hopkins School of Public Health, Baltimore, Maryland, United States of America, 4 Research
a1111111111 Unit, Asociación Benéfica Prisma, Iquitos, Peru, 5 Oregon Neurosurgery, Eugene, Oregon, United States of
America, 6 Division of Integrative Systems Medicine and Digestive Diseases, Department of Surgery and
Cancer, Imperial College London, London, United Kingdom

* [email protected]

OPEN ACCESS

Citation: Colston JM, Peñataro Yori P, Moulton LH,

Paredes Olortegui M, Kosek PS, Rengifo Trigoso D,
Abstract
et al. (2019) Penalized regression models to select
biomarkers of environmental enteric dysfunction
Environmental enteric dysfunction (EED) is associated with chronic undernutrition. Efforts
associated with linear growth acquisition in a to identify minimally invasive biomarkers of EED reveal an expanding number of candidate
Peruvian birth cohort. PLoS Negl Trop Dis 13(11): analytes. An analytic strategy is reported to select among candidate biomarkers and sys-
e0007851. https://doi.org/10.1371/journal.
tematically express the strength of each marker’s association with linear growth in infancy
pntd.0007851
and early childhood. 180 analytes were quantified in fecal, urine and plasma samples taken
Editor: Andrew S. Azman, Johns Hopkins
at 7, 15 and 24 months of age from 258 subjects in a birth cohort in Peru. Treating the sub-
Bloomberg School of Public Health, UNITED
STATES jects’ length-for-age Z-score (LAZ-score) over a 2-month lag as the outcome, penalized lin-
ear regression models with different shrinkage methods were fitted to determine the best-
Received: July 5, 2019
fitting subset. These were then included with covariates in linear regression models to obtain
Accepted: October 16, 2019
estimates of each biomarker’s adjusted effect on growth. Transferrin had the largest and
Published: November 15, 2019 most statistically significant adjusted effect on short-term linear growth as measured by
Copyright: © 2019 Colston et al. This is an open LAZ-score–a coefficient value of 0.50 (0.24, 0.75) for each log2 increase in plasma transfer-
access article distributed under the terms of the rin concentration. Other biomarkers with large effect size estimates included adiponectin,
Creative Commons Attribution License, which
arginine, growth hormone, proline and serum amyloid P-component. The selected subset
permits unrestricted use, distribution, and
reproduction in any medium, provided the original explained up to 23.0% of the variability in LAZ-score. Penalized regression modeling
author and source are credited. approaches can be used to select subsets from large panels of candidate biomarkers of
Data Availability Statement: All relevant data are EED. There is a need to systematically express the strength of association of biomarkers
within the manuscript and its Supporting with linear growth or other outcomes to compare results across studies.
Information files.

Funding: MNK received grants OPP1066146 and

OPP1152146 from the Bill & Melinda Gates
Foundation (www.gatesfoundation.org). The
funders had no role in study design, data collection

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 1 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

and analysis, decision to publish, or preparation of

the manuscript. Author summary
Competing interests: The authors have declared Childhood undernutrition is widespread throughout the world and has severe, long-last-
that no competing interests exist.
ing health impacts. Substances measured in blood, urine and stool could be used as bio-
markers to identify children undergoing growth failure before these impacts occur.
However, it is not yet known which of the many markers that can be identified are accu-
rate and clinically useful predictors of poor growth in infants and children. This study
used a large number of candidate biomarkers of immune activation, metabolism and hor-
mones and applied statistical methods to narrow them down from 110 different sub-
stances, to the 36 best predictors of growth in 258 Peruvian infants. It also estimated how
large the effect of each of these markers was on height two months later. The biomarker
with the largest effect was transferrin, a glycoprotein that can be measured in blood sam-
ples. 15-month old children with elevated transferrin were around two thirds of a centi-
meter taller on average at 17 months than those with low levels. Transferrin and other
proteins, glycoproteins, hormones and antibodies that this study identified, can be mea-
sured easily and affordably in standard laboratories making them feasible to be used
broadly as prognostic markers as part of child health and nutrition programs in under-
resourced settings.

Introduction
Chronic undernutrition affects around one in three children under age five, rendering them
susceptible to prolonged and more severe infections and putting them at increased risk of mor-
tality [1]. Growth faltering in undernourished children begins to accrue early in life, is gener-
ally irreversible and leads to chronic sequelae such as impaired cognitive development and
short stature that last into adulthood impeding economic productivity and increasing the risk
of low birthweight in offspring [2]. Many evidence-based interventions targeting infant growth
demonstrate only modest improvements in outcomes in effectiveness trials [3], a gap that, it is
increasingly suspected, may be partially explained by a phenotype of intestinal abnormalities
known as environmental enteric dysfunction (EED) [4], which is gaining recognition as a
neglected disease [5]. According to the EED hypothesis, concurrent exposures to multiple
enteric pathogens in already undernourished children cause cumulative damage to their guts’
surface, increasing its permeability to microbes and large molecules, causing systemic inflam-
mation and impairing uptake and utilization of nutrients [6–8], which in turn leads to sub-
optimal growth [9].
Studying the impact of EED is challenging. Gold standard diagnostic tests for other enter-
opathies, such as celiac and Crohn’s disease, include endoscopy and gut biopsy, invasive and
demanding procedures that cannot feasibly be deployed in resource-constrained settings or to
assess disease burden at the population level [10]. For this reason, there is considerable interest
in identifying and validating biomarkers of EED that can be used as surrogate endpoints in
population-based studies and for evaluating nutrition and hygiene interventions [11]. The
most widely adopted biomarkers of EED use saccharide-based permeability assays like the lac-
tulose/mannitol test [12]. However, such tests, while non-invasive, have well-documented lim-
itations to their use in EED-endemic populations, taking hours to administer, requiring
samples to be shipped to well-equipped facilities which makes them cumbersome, expensive
and impractical for screening and randomization for intervention trials [13]. Several fecal bio-
markers, such as alpha-1-antitrypsin, myeloperoxidase and neopterin, have been shown to
have complex associations with growth outcomes [9,11], while certain plasma biomarkers

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 2 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

show correlations with suboptimal growth, including the amino acid tryptophan and its ratio
to its derivative, kynurenine [14].
Recently developed methods allow for quantifying large panels of soluble analytes in blood
that relate to inflammation or immune status [15], however there is a lack of consensus about
how to select the most important markers from among these panels and quantify their associa-
tion and explanatory power with respect to specific disease outcomes relevant to EED (growth,
cognitive function, immune activation, intestinal permeability, nutrient bioavailability, and
hormones that alter growth and metabolism) [11,16]. Machine learning approaches have been
used in biomarker analyses to identify best subsets of predictors from among large databases
of candidate markers [16–18]. More specifically, penalized regression methods estimate coeffi-
cient values for modeled variables, while applying different penalties to those that overly
increase model complexity relative to improving goodness of fit, assigning such variables a
coefficient value of (“shrunk” to) zero. Those variables that are assigned non-zero coefficients
can be interpreted as belonging to the subset that best predicts the outcome. Although these
methods do not themselves report standard errors or adjust for within-cluster correlation in
longitudinal data, the selected subsets can be included in more traditional multivariate regres-
sion models once identified and the effect size described by conventional methods.
The objective of this study was to identify clinically relevant biomarkers of the precursors of
EED that can inform intervention early in the disease process. To this end, penalized regres-
sion approaches for variable subset selection were applied to a large panel of candidate bio-
markers measured in a cohort of Peruvian infants to identify the optimal subset that are most
predictive of nutritional status (length-for-age Z-score–LAZ-score) over a two-month lag.

Methods
Ethical approval and consent to participate
Ethical approval for MAL-ED was given by the Johns Hopkins Institutional Review Board as
well as the Ethics Committee of Asociacion Benefica PRISMA, and the Regional Health
Department of Loreto. Written informed consent was obtained from the caregiver of every
participating child.

Study population
A cohort of 303 infants was enrolled between December 2009 and February 2012 from Santa
Clara de Nanay, a peri-urban community located 15 km from the city of Iquitos, Peru, a study
setting that has been described in detail elsewhere [19]. Singleton births from a selected geo-
graphic area were enrolled within 17 days of birth provided they had no recognized congenital
defects and weighed >2.4 kg at birth [20] and were followed up until 5 years of age. Daily data
relating to infant feeding were ascertained by caregiver report from twice-weekly household
visits from age 0 to 24 months, while anthropometric data and biological samples were col-
lected during monthly assessments according to pre-established schedules [20].

Outcome variable
The outcome of interest in this analysis was the subjects’ LAZ-score, a widely used measure of
nutritional status and attained statural growth [21–23] that were calculated using WHO
Anthro version 3.2.2. Anthropometric assessments were carried out at monthly intervals
counted from the subjects’ birth dates from enrolment until 5 years of age. During these assess-
ments, infants’ lengths were measured on marked platforms with a sliding footboard employ-
ing quality control measures that have been described elsewhere [23]. The LAZ-score was

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 3 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

treated as a continuous variable. Its distribution in this study population has also been
described elsewhere [11].

Exposure variables
The primary exposure variables were 180 time-varying candidate fecal, urinary and plasma
biomarkers of EED compiled from the following panels (biomarker names, abbreviations and
units are listed in the supporting information):
1. Three overlapping panels of in-house quantitative, multiplexed immunoassays of cytokines,
chemokines, hormones and other regulators of metabolism and growth [24], each run at
the Myriad RBM laboratories (Austin, TX) on a separate subset of blood samples from the
cohort including:

a. 86 analytes from 20 samples taken at age 7 months from a sub-sample of subjects and
run in June 2013. These 20 subjects (10 cases and 10 controls) were selected for more
expansive testing to examine extremes in growth in this setting over the target period
between 6–15 months when exclusive breastfeeding is no longer the optimal feeding
practice. Cases (positive deviants) were those subjects who grew by >0.77 LAZ, while
controls (negative deviants) were selected from those subjects who experienced a change
in LAZ of <0.25 over the 8-month follow-up period. This sub-sample was selected for a
separate study to compare extremes in growth in this setting over the same period. At
the age of 7 months, both cases and controls had equal LAZ.
b. 49 analytes from 178 samples mostly taken at the target age of 24 months (though with a
small number taken at 7, 15, 25 and 26 months) run in May 2014.
c. 59 analytes from 443 samples taken at the target ages of 7 and 15 months (though with a
small number taken at 8–9 or 16–18 months) run in January 2015.
2. 9 chemokine and 9 proinflammatory assays run on 596 of the same blood samples as 1 a-c
at a laboratory at Johns Hopkins University (Baltimore, MD) in 2013–2014.
3. The amino acids citrulline and tryptophan and the latter’s metabolite kynurenine (umol/L)
quantified in 640 of the same blood samples by liquid chromatography-mass spectrometry
(LCMS) in the Oregon Analytics laboratory in 2015 [14].
4. 51 other biogenic amines quantified in 464 of the same blood samples by LCMS at a labora-
tory in Imperial College London in 2017 [25].
5. Several plasma analytes measured in the same blood samples as part of the MAL-ED proto-
col, including Alpha-1-acid glycoprotein (AGP—mg/dl, measured by radioimmune diffu-
sion assay in 618 samples), Insulin-like growth factor (IGF) 1 and IFG-binding protein 3
(IGFBP-3—measured by enzyme-linked immunosorbent assay (ELISA) in 566 and 597
samples respectively) and hemoglobin (g/dL, measured by Hemocue).
6. Three fecal biomarkers—alpha-1-antitrypsin (AAT–mg/g), myeloperoxidase (MPO–ng/
mL) and neopterin (NEO–nmol/L)—measured by ELISA tests of stool samples collected
from the infants at monthly intervals [26].
7. 5 urinary biomarkers calculated from lactulose to mannitol recovery tests of intestinal per-
meability performed on urine samples collected at 3, 6, 9 and 15 months of age.
Table 1 shows the number of biological samples and analytes available in each panel by age
of the subjects. In addition, the following variables were included as potential confounders:

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 4 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

Table 1. Number of biological samples and analytes available in each panel included in the study by age at which they were taken.
Panel number
1 2 3 4 5 6 7
a. b. c. AGP IGF-1 IGFBP-3 Hb
Age in months 6 0 0 0 0 0 0 0 0 0 0 174 267
7 20 5 211 210 226 148 236 175 202 340 262 2
8 0 0 2 2 7 2 2 2 1 7 264 1
9 0 0 2 2 3 2 2 1 2 6 175 247
10 0 0 0 0 0 0 0 0 0 0 257 3
11 0 0 0 0 0 0 0 0 0 0 248 0
12 0 0 0 0 0 0 0 0 0 0 253 0
13 0 0 0 0 0 0 0 0 0 0 209 0
14 0 0 0 0 0 0 0 0 0 0 203 0
15 0 6 211 189 209 156 200 179 183 355 177 226
16 0 0 13 10 11 12 12 12 11 14 213 1
17 0 0 3 2 3 2 2 2 3 5 214 2
18 0 0 1 1 1 1 1 1 1 2 226 0
19 0 0 0 0 0 0 0 0 0 0 221 0
20 0 0 0 0 0 0 0 0 0 0 218 0
21 0 0 0 0 0 0 0 0 0 0 213 0
22 0 0 0 0 0 0 0 0 0 0 206 0
23 0 0 0 0 0 0 0 0 0 0 197 0
24 0 167 0 167 167 129 154 181 182 304 182 180
25 0 9 0 9 9 8 8 9 8 13 52 4
26 0 4 0 4 4 4 1 4 4 4 43 1
Total 20 191 443 596 640 464 618 566 597 1,050 4,207 934
Analytes 86 49 59 18 3 53 1 1 1 1 3 5
https://doi.org/10.1371/journal.pntd.0007851.t001

infants’ sex, birthweight, breastfeeding status on the previous day (a time-varying categorical
variable with four categories—“exclusively breastfed”, “partially breastfed”, “predominantly
breastfed” and “not breastfed”), age in whole months (modeled using linear and quadratic
terms) and mother’s height at the time of birth.

Statistical analysis
The fecal and urinary samples were matched to the plasma biomarker values that were closest
in age and those that were not matched to any blood sample were excluded from the analysis.
Exposure values were lagged by two months so that the analysis assessed the association
between the subjects’ LAZ-score at month of age j and the exposures measured at age j-2
months. A 2-month lag was chosen because it is a length of time at which the impacts on a
child’s growth of interventions such as steroids [27], chemotherapy [28] or treatment for
severe acute malnutrition [29] become manifest, and therefore offers a feasible time window
for clinical intervention and in which to reproducibly detect meaningful changes in ponderal
growth associated with important physiologic determinants. Two months has also been dem-
onstrated to be optimal for predicting future growth trajectory using fecal biomarkers [11]. All
biomarkers were log-transformed with base 2. Because numerous biomarkers were either only
available for samples collected at 24 months of age, or only for those collected around 7 and
15 months of age, the following analyses were performed on two subsets of the full biomarker
database:

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 5 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

1. 7–15-month database–This excluded the samples in panel 1.b and any samples from panels
2–7 that were taken at �24 months of age, resulting in 461 observations and 110
biomarkers.
2. 7–24-month database–This included the 24-month samples but only for those biomarkers
that were included in both panels 1.b and 1.c as well as panels 1. a and 2–7, resulting in 639
observations and 80 biomarkers.

Missing data. Non-detectable biomarker values, for which the analyte concentration was
p
below the lower limit of quantification (LLOQ), were substituted with LLOQ / 2 [30]. No
standard equivalent approaches exist for substituting values that are above the upper limit of
quantification (ULOQ), however this only affected a small number of values for biomarkers in
panel 4 which were treated as missing values. Almost all biomarkers and subjects had some
number of missing values. Biomarkers for which more than 40% of the original values were
missing were excluded from the imputation and further analysis, as were variables with fewer
than 25 unique values within the detectable range. Observations that had missing values for
more than 40% of the remaining biomarkers were excluded from the analysis. A small number
of missing length measurements (n = 19, 3.0% of total) were linearly interpolated and extrapo-
lated based on the actual or target date of assessment before calculating LAZ-scores. For time-
fixed baseline variables (birth weight and maternal height), the small number of missing values
were substituted with the sample mean of that variable. All other missing values of the bio-
marker exposures were imputed using multivariate normal regression (MVN) with an iterative
Monte Carlo method to accommodate the arbitrary missing-value patterns of the continuous
variables [31]. Missing values–of which there were 5,279 (11.1%) in the 7-15-month database
and 4,508 (10.6%) in the 7–24-month database—were substituted with the average of the
imputed values from 10 MVN imputations. The kynurenine/tryptophan (K/T ratio) and lactu-
lose/mannitol ratios were excluded from imputation and recalculated after from their compo-
nent biomarkers.
Variable selection. The retained biomarkers were included in penalized linear regression
models with three different shrinkage methods that have been used in other studies of EED
biomarkers—Adaptive LASSO (Least Absolute Shrinkage and Selection Operator), Minimax
Concave Penalty (MCP) and Smoothly Clipped Absolute Deviation (SCAD) penalties [16,17]–
with values for the tuning parameter λ determined through 10-fold cross validation. For each
model, the variables assigned non-zero coefficients were treated as the optimal, best-predicting
subset and the subset for the method that yielded the lowest cross-validation error (calculated
from the mean-squared error or deviation from the fitted mean) was retained in a final multi-
variable model.
Effect modeling. Regression models were fitted with robust variance estimation to allow
for intra-subject correlation first for each of the candidate biomarkers separately (adjusting for
the a priori-selected non-biomarker covariates) in order to report their independent effects
and statistical significance and then for a multi-variable model that included all biomarkers
selected for the best-fitting subset, to estimate the adjusted effect of each in the presence of the
others and their combined effect on LAZ-score. To account for the false discovery rate (FDR)
due to the large number of comparisons, p-values from the separately modeled biomarkers
were compared visually in scatterplots with their corresponding q-values (a measure of signifi-
cance in terms of the FDR [32] calculated using the method proposed by Simes [33]) and with
a Bonferroni corrected α value calculated from the number of comparisons. The effect mea-
sures from the single-biomarker and adjusted subset models were visualized using forest plots.
For the biomarkers included in the final, multi-variable models, the coefficient estimates were

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 6 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

reported along with the difference in a child’s height predicted by the model between subjects
at the 25th and the 75th percentile of each included biomarker’s distribution at the age of the
final included sample (15 or 24 months, depending on which database was used) and holding
all other included biomarkers at their mean values and based on the standard deviation in
height at that age reported in the WHO child growth standards [21]. The R2 statistic for the
final subset model was reported along with the partial R2 for all included biomarker terms as
an estimate of the proportion of the total variability in the outcome that was explained by the
selected biomarker subset. Results from the final models were compared with those obtained
from adjusting for LAZ-score measured contemporaneously with the biomarker (in place of
the other covariates), in order to compare the prognostic potential of the final biomarker sub-
set in predicting future growth relative to a natural and existing alternative, namely attained
LAZ-score. The potential for non-linear relationships between biomarkers in the final subsets
and LAZ-score was assessed by generating nonparametric smooth plots and by applying a
multivariate spline model-selection algorithm to the final models. Finally, as a validation exer-
cise, associations between each of three of the most important biomarkers (expressed per stan-
dard deviation [SD]) and changes in LAZ over increasing lag-lengths of 1–10 months
adjusting for contemporaneous LAZ were plotted to assess the performance using an existing
method that has previously been used for tryptophan and citrulline and compared to a com-
parator biomarker of a known endocrinologic agent–Insulin-like growth factor 1 (IGF-1), rep-
licating the methodology of Kosek and colleagues [14]. Analyses were carried out using Stata
15.1 [34] and R 3.6.1.

Results
Summary statistics of the distributions of the 180 candidate biomarkers and whether they met
the criteria for inclusion in further analysis are presented in S1 Table in the supporting infor-
mation. A participant flowchart is provided as S1 Fig (supporting information). Before apply-
ing exclusion criteria, 639 observations were available for 258 of the 303 enrolled subjects for
whom blood samples were available relating to 180 biomarkers. 23 of the biomarkers were
only available in the case control panel (panel 1a.) and so were excluded from further analysis
for only having 20 available observations. A further 47 biomarkers were excluded from the 7-
15-month database either because more than 40% of their values were missing, fewer than 25
were within the detectable range, they were only available at 24 months of age (panel 1c.) or
some combination of these. 77 biomarkers were excluded from the 7-24-month database due
to missingness, detectability, or because they did not have values available at 24 months. Over-
all, 110 biomarkers were retained for analysis in the 7-15-month database, and 80 in the 7-
24-month.
Table 2 shows the number of biomarkers selected (assigned non-zero coefficients) and the
cross-validation error and R2 values for the three penalized regression models fitted on each of
the two biomarker databases. MCP selected the smallest subset of biomarkers when fitted to
the 7-15-month, but not the 7-24-month database, for which adaptive LASSO selected the
smallest. For both databases, the SCAD penalty resulted in the largest subset, the highest cross-
validated R2 and the lowest cross-validation error (jointly with MCP in the 7–24 month
model) and was chosen as the subset for subsequent analyses.
Fig 1 shows the coefficient estimates from the linear regression models of single biomark-
ers, for the subsets of multiple biomarkers selected by the three penalized regression methods
and from fitting a final multi-variable linear regression model to the SCAD-selected subset–
the penalty with the lowest cross-validation error—adjusting for covariates. For 82 of the 110
biomarkers in the 7-15-month database, the single biomarker model predicted a negative

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 7 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

Table 2. Number of biomarkers selected (assigned non-zero coefficients) and cross-validation error for three penalized regression models fitted on two biomarker
databases.
7–15 months 7–24 months
Adaptive LASSO MCP SCAD Adaptive LASSO MCP SCAD
Biomarkers selected 17 8 23 5 22 25
Cross-validation error 0.84 0.84 0.82 0.85 0.78 0.78
Cross-validated R-squared 0.06 0.05 0.08 0.10 0.10 0.10
https://doi.org/10.1371/journal.pntd.0007851.t002

association with the outcome, compared with 28 for which a positive association was pre-
dicted. In 17 of these models, the estimate was statistically significant at the uncorrected α =
0.05 level. 42 of the 80 biomarkers in the 7-24-month database had a negative association with
the outcome in the single biomarker models, 38 had a positive association, and 12 had statisti-
cally significant estimates. No fecal or urinary biomarkers were included in the final subsets
selected by adaptive LASSO, although in both the 7–15 and 7-24-month models, the SCAD
and MCP penalties assigned small, non-zero coefficients to fecal MPO and SCAD also selected
urinary lactulose.
In both databases, just 5 biomarkers were selected by all three penalties. In the 7-15-month
models, these were hemoglobin, Immunoglobulin A (IgA), Insulin-like growth factor-binding
protein 3 (IGFBP-3), Pulmonary and Activation-Regulated Chemokine (PARC) and Thyroid-
Stimulating Hormone (TSH), while in the 7-24-month models these included adiponectin and
IgM instead of IgA and TSH. In both databases, all biomarkers selected by MCP were also
selected by SCAD and in the 7-24-month database all biomarkers selected by adaptive LASSO
were also selected by the other two penalties. SCAD selected 5 biomarkers in the 7-15-months
and 3 in the 7-24-month database that were not included in either of the other two subsets,
however only one of these–growth hormone (GH)–was significant in the final model.
Fig 2 plots the p-values from the separately modeled biomarkers against their correspond-
ing q-values with lines representing the Bonferroni corrected α values to assess their signifi-
cance after adjusting for the FDR. For both databases, only adiponectin retained statistical
significant at the Bonferroni corrected α levels, while a small number of other biomarkers–Fer-
ritin (FRTN), IGF-1, IGFBP-3 and Serum Amyloid P-Component (SAP) in both databases,
GH and aspartic acid for the 7-15-month data and PARC for the 7-24-month–had q-values
below the less conservative threshold of q<0.1.
Table 3 presents the coefficient estimates from the final 7-15-month and 7-24-month linear
regression models for a 1 log2 increase of each of the biomarkers selected by SCAD along with
the difference in child’s height predicted for children aged 17 and 26 months respectively at
the 25th and 75th percentile of the biomarker distribution (holding all other included biomark-
ers at their sample mean). The 36 selected biomarkers include numerous amino acids, chemo-
kines, hormones, glycoproteins and proteins along with two antibodies, three apolipoproteins,
the enzyme myeloperoxidase, the sugar lactulose and 5-OH-Indole-3-acetic Acid (5-HIAA),
the metabolite of serotonin. Thirteen biomarkers were included in both final models, while 11
were only included in the 7-15-month model and 12 only in the 7-24-month model.
The iron-transporting glycoprotein transferrin had the largest effect size in the 7-15-month
model both in terms of its estimated coefficient–a highly statistically significant 0.50 (0.24,
0.75) increase in the predicted LAZ-score–and the height difference predicted–a 17-month-
old child at the 75th percentile of plasma transferrin concentration being two thirds of a centi-
meter taller than one at the 25th. Hemoglobin had the second largest absolute coefficient value
in the 7-15-month model–a slightly significant 0.47 (0.02, 0.93)–but the second largest differ-
ence in height was predicted by SAP—a child at the 3rd quartile of its distribution predicted to

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 8 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 9 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

Fig 1. Forest plots of coefficient estimates and 95% confidence intervals from linear regression models of single biomarkers, for subsets of
multiple biomarkers selected by the three penalized regression methods and from a final multi-variable linear regression model of the subset
with the lowest cross-validation error adjusting for covariates.
https://doi.org/10.1371/journal.pntd.0007851.g001

be 0.65 cm shorter than one at the 1st quartile–which also had a highly statistically significant
coefficient estimate. Other biomarkers for which the 7-15-month model predicted large and
statistically significant negative effects include the hormones adiponectin and GH–predicting
respectively around a -0.4cm and a -0.28cm height difference–and apolipoprotein (Apo)
C-I—-0.3cm–while AGP had a slightly statistically significant positive effect.
Several biomarkers that had large effect sizes in the 7-15-months model–transferrin, Apo
C-I and GH- were not included in the 7-24-month database, due to no values being available
at 24 months of age. Instead, in that model, while hemoglobin again had the largest coefficient
estimate–a non-significant 0.44 (-0.03, 0.91)–SAP predicted the largest difference in height
between the extremes of the interquartile range of the analyte’s distribution at 24 months–
26-month-old children with high SAP concentration at 24-months a predicted 0.79cm shorter
than their low SAP counterparts–the next largest being the chemokines Interleukin-8 (IL-8)–
0.63cm taller–and adiponectin– 0.54cm shorter–the latter having a highly statistically signifi-
cant effect estimate. Proline, arginine, tryptophan and SHBG also all had slightly statistically
significant coefficient estimates and predicted among the largest height differences.
The final 7-15-month model explained 43.0% of the variance in the LAZ-score according to
the R2 statistics, with 23.0% of the variance explained solely by the selected subset of biomark-
ers (the partial R2 statistic excluding the non-biomarker covariates). The equivalent propor-
tions for the final 7-24-month model were 39.6% and 17.7% respectively. S2 Table in the
supporting information show the equivalent results when the non-biomarker covariates were
replaced in the final models with contemporaneous LAZ-score to adjust for attained growth.
In the presence of this variable, many of the effect size estimates decreased in magnitude and

Fig 2. Scatterplot comparing the p-values from the separately modeled biomarkers to their corresponding q-values calculated using the method proposed by
Simes’ method [33] and to the Bonferroni corrected α values (represented by the dashed lines). Biomarkers for which q<0.1 are labeled.
https://doi.org/10.1371/journal.pntd.0007851.g002

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 10 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

Table 3. Coefficient estimates (with 95% confidence intervals) from linear regression models for biomarkers selected by SCAD along with the predicted difference
in child’s height 2 months after the last sample for children at the 25th and 75th percentile of the biomarker distribution.
Biomarker 7 & 15 months 7, 15 & 24 months
Coefficient—LAZ Predicted height difference (cm) at Coefficient—LAZ Predicted height difference (cm) at
score 17 months score 26 months
5-OH-Indole-3-acetic Acid (5-HIAA) - - -0.05 -0.16
(-0.11, 0.00)
Alpha-2-Macroglobulin (A2Macro) -0.07 -0.10 - -
(-0.25, 0.11)
Alpha-amino-n-butyric acid (AABA) -0.06 -0.15 - -
(-0.18, 0.06)
Adiponectin -0.26 -0.40 -0.29 -0.54
(-0.42, -0.10) (-0.47, -0.12)
alpha-1-acid glycoprotein (AGP) 0.20 0.33 0.07 0.14
(0.05, 0.36) (-0.09, 0.23)
Apolipoprotein B (Apo B) - - 0.06 0.13
(-0.11, 0.23)
Apolipoprotein C-I (Apo C-I) -0.22 -0.30 - -
(-0.43, -0.01)
Apolipoprotein D (Apo D) - - -0.08 -0.12
(-0.26, 0.11)
Arginine 0.18 0.39 0.17 0.46
(-0.00, 0.37) (0.00, 0.34)
Beta-amino-iso-butyric acid (BABA) - - 0.06 0.38
(-0.03, 0.15)
Citrulline -0.15 -0.22 -0.14 -0.24
(-0.32, 0.03) (-0.33, 0.06)
Eotaxin-3 - - 0.06 0.11
(-0.04, 0.16)
Fecal Myeloperoxidase (MPO) 0.03 0.19 0.03 0.20
(-0.01, 0.08) (-0.02, 0.07)
Growth Hormone (GH) -0.07 -0.28 - -
(-0.12, -0.01)
Hemoglobin 0.47 0.30 0.44 0.31
(0.02, 0.93) (-0.03, 0.91)
Homoserine -0.05 -0.16 - -
(-0.12, 0.01)
Immunoglobulin A (IgA) -0.12 -0.23 - -
(-0.27, 0.03)
Immunoglobulin M (IgM) - - -0.03 -0.07
(-0.16, 0.11)
Insulin-like growth factor-binding protein 3 0.19 0.25 0.18 0.28
(IGFBP-3) (-0.06, 0.43) (-0.02, 0.39)
Interleukin-8 (IL-8) chemokine - - 0.17 0.63
(0.07, 0.27)
Lactulose -0.05 -0.24 - -
(-0.11, 0.01)
Leptin 0.02 0.09 0.06 0.27
(-0.07, 0.11) (-0.02, 0.14)
Lysine 244 - - -0.07 -0.21
(-0.15, 0.01)
Monocyte Chemotactic Protein 4 (MCP-4) - - -0.07 -0.25
(-0.21, 0.07)
Myoglobin - - 0.02 0.06
(-0.08, 0.12)
(Continued )

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 11 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

Table 3. (Continued)

Biomarker 7 & 15 months 7, 15 & 24 months

Coefficient—LAZ Predicted height difference (cm) at Coefficient—LAZ Predicted height difference (cm) at
score 17 months score 26 months
Pulmonary and Activation-Regulated -0.02 -0.04 -0.14 -0.28
Chemokine (PARC) (-0.19, 0.15) (-0.31, 0.03)
Proline -0.21 -0.42 -0.24 -0.50
(-0.44, 0.02) (-0.44, -0.05)
Serum Amyloid P-Component (SAP) -0.28 -0.65 -0.29 -0.79
(-0.42, -0.14) (-0.47, -0.12)
Sarcosine 0.00 0.01 - -
(-0.09, 0.09)
Sex Hormone-Binding Globulin (SHBG) -0.11 -0.33 -0.12 -0.42
(-0.23, 0.01) (-0.22, -0.01)
Thymus and activation regulated chemokine - - -0.01 -0.05
(TARC) (-0.11, 0.09)
Thyroxine-Binding Globulin (TBG) - - 0.28 0.36
(-0.01, 0.56)
Transferrin 0.50 0.66 - -
(0.24, 0.75)
Tryptophan 0.17 0.29 0.23 0.44
(-0.03, 0.37) (0.06, 0.40)
Thyroid-Stimulating Hormone (TSH) -0.07 -0.16 - -
(-0.17, 0.03)
von Willebrand Factor (vWF) 0.07 0.23 - -
(-0.01, 0.14)
https://doi.org/10.1371/journal.pntd.0007851.t003

statistical significance considerably including transferrin and GH in the 7-15-month model,

tryptophan and SHBG in the 7-24-month model and adiponectin and SAP in both models.
Several biomarkers did increase in statistical significance upon adjustment for attained growth
however, including Alpha-2-Macroglobulin (A2Macro), fecal MPO, tryptophan and TSH
in the 7-15-month and proline and hemoglobin in the 7-24-month models. Adjustment for
baseline LAZ-score also greatly increased the proportion of the variability explained by the
models—R2 statistics of 83.7% and 84.3% for the 7-15-month and 7-24-month models respec-
tively–but decreased the proportion explained by the biomarker subsets– 9.6% and 7.3%
respectively—demonstrating that growth already attained has far more explanatory power for
modeling short-term future growth than any combination of biomarkers.
Numerous biomarkers, including Eotaxin-3, citrulline, myoglobin, lactulose, and SHBG,
exhibited evidence of having non-linear relationships with the outcome when visualized in
polynomial smooth plots (S2–S6 Figs respectively in the supporting information). When a
multivariate spline model-selection algorithm was run on each of the two final biomarker sub-
sets, none of the biomarkers improved the model when represented by multiple cubic splines
relative to linear terms with the exceptions of proline in the 7-15-month model (4 degrees of
freedom) and Thymus and activation regulated chemokine (TARC) and Monocyte Chemotac-
tic Protein 4 (MCP-4) (2 degrees of freedom each) in the 7-24-month model (results not
reported).
Fig 3 shows the results of the validation exercise in which a previously published methodol-
ogy was replicated using three biomarkers from the final subset identified here along with
IGF-1 as a comparator. This analysis treated the difference in LAZ-score (ΔLAZ) over time-
windows of increasing length as the outcome, standard deviations of the biomarkers as expo-
sures and adjusted for baseline LAZ, as well as the other covariates. Adiponectin, which had

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 12 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

Fig 3. Associations between concentrations of four plasma biomarkers (per standard deviation of their log2 transformed values) and
differences in LAZ-score (ΔLAZ) over time windows of increasing length, from models adjusting for baseline LAZ, age, and sex.
https://doi.org/10.1371/journal.pntd.0007851.g003

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 13 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

previously exhibited a large and highly statistically significant association with nutritional sta-
tus showed no obvious trend after adjustment for attained growth, while IGF-1 and, most
markedly, transferrin showed large and statistically significant associations with changes in
LAZ-score over longer time windows of 5–10 months.

Discussion
Analytical techniques such as multiplex immunoassays and mass spectrometry are increasingly
being used in human studies to enable the quantification of ever more diverse and extensive
panels of analytes in biological samples, many of which have biological functions that have yet
to be fully characterized. At the same time, advanced statistical learning methods have
emerged that can be used to identify patterns in large datasets. This study brings together these
two developments and applies them to an issue that has received growing attention in recent
years but has yet to be fully resolved–identifying prognostic biomarkers of EED that can pre-
dict future linear growth over time windows relevant to clinical intervention. In a birth cohort
recruited from a low-resource setting in Peru, this study reports the distributions of 180 candi-
date biomarkers in fecal, urinary and plasma samples, of which 110 met the criteria for inclu-
sion in variable-subsetting penalized regression models–the largest number of markers ever
considered in a study of this nature.
The final subsets selected by SCAD penalty included numerous biomarkers that previous
studies have implicated as potential predictors of linear growth and markers of gut function.
The essential amino acid tryptophan has previously shown promise as a prognostic indicator
of EED due to its role in normal infant growth and its hypothesized correlation with indolea-
mine 2,3-dioxygenase 1 (IDO1) activity in states of chronic low-grade endotoxin exposure
[14]. However, while tryptophan was selected by the majority of the penalized regression mod-
els, and its association with LAZ-score was statistically significant in the 7-24-month final
models it was not among the biomarkers most predictive of differences in height. A positive
association between plasma tryptophan concentration and a 6-month change in LAZ-score
has already been reported in this cohort and a similar one in Tanzania and separately in one in
Northeast Brazil with effect sizes comparable to that of the final model here [14,35]. Immuno-
globulin A (IgA), which was retained in the final 7-15-month model, had a small, non-signifi-
cant, negative effect size consistent with that observed for IgA anti-LPS antibody also in the
Brazil cohort [35].
For some other biomarkers in the subsets, evidence in previous literature on EED is more
scant though known mechanisms nonetheless exist through which they might plausibly track
nutritional status. Most obvious of these is hemoglobin, long the gold standard marker of
severe anemia and therefore of its attendant delaying effects on growth and development [36].
Analysis of data from the 8-site study to which the cohort described here contributed found an
association (though weaker and less significant than those found here) between hemoglobin
and LAZ-score at age 5 years [37], while other studies of EED have adjusted for hemoglobin as
a potential confounder [38,39]. Low levels of plasma transferrin are found during protein-
energy malnutrition [40]. Adiponectin is an appetite-regulating hormone that promotes satiety
and therefore may inhibit food intake, which may explain its negative association with growth
[41,42]. While elevated levels of circulating adiponectin have a known negative association
with obesity [43], its role in child growth is unclear, and among twins this adipokine had a pos-
itive association with birthweight-adjusted LAZ-score (counter to the negative one reported
here) [42]. Leptin and the serum leptin-adiponectin ratio were found to be associated with
stunting in Bangladeshi children and increased in this group following food supplementation
[38]. The positive association between serum arginine concentrations and nutritional status is

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 14 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

consistent with findings from Malawi, though the same study failed to find a significant associ-
ation with proline, which was one of the more predictive of the biomarkers in these results
[44].
While the SCAD-selected subset was used for the final models due to its yielding the low
cross-validated error and explaining a larger proportion of the variance, it is notable that this
penalty did select several biomarkers that had small non-significant effect estimates and did
not select several biomarkers, which had statistically significant single biomarker effect sizes
and known associations with nutritional outcomes (such as IGF-1 and ferritin). Though
SCAD has been used in numerous studies of EED biomarkers [16–18], these findings do sug-
gest that this penalty lacks both sensitivity and specificity when applied to large panels.
For other biomarkers in the subsets, the functions or pathways through which they might
impact growth are as yet unclear, which demonstrates the hypothesis-generating potential of
this approach. SHBG is of interest in biomarker research for its association at low levels with
type-II diabetes and metabolic syndrome but, although elevated SHBG is seen following
weight loss, this glycoprotein has not previously been considered as a prognostic marker of
growth faltering [45]. Though known for its association with amyloidosis, SAP is also involved
in the humoral innate immune system’s response to infections and might plausibly lie on the
pathway connecting enteric pathogen infection to growth deficits that is specific to the EED
hypothesis [46–48]. TBG, responsible for binding the thyroid hormones thyroxine and triiodo-
thyronine in the blood down, which downregulate the activity of hormones that stimulate met-
abolic rate and may influence the regulation of skeletal growth [49,50].
C-Reactive Protein (CRP), which multiple previous studies have found to be a promising
biomarker [17,51], was not selected despite having a statistically significant, though small, neg-
ative effect in the single biomarker 7–24 months model. The fact that CRP is inversely related
to Fetuin-A [52] and, like SAP, is a calcium-dependent ligand binding plasma protein [46]
may mean that the presence of the latter protein in the final model fully accounted for any
effect of CRP. The three fecal biomarkers and the urinary lactulose/mannitol ratio (along with
the other four urinary markers) have shown clinical potential in previous studies [11,53] but in
this analysis were not significant in any of the single-biomarker or final models. It may be the
case that restricting the data to assessments at just 2–3 time points meant that the analysis was
underpowered to detect the true but relatively small effects of these substances [11]. Citrulline,
which has shown promise in previous studies [35], was not significant in either single bio-
marker model, and was selected but not significant in the final models.
Although ferritin, the body’s stored form of iron, has been implicated previously [17,51]
and was significant in the single biomarker model, it was not selected here for either final
model. This may be because its association with growth is mediated by the stronger and more
statistically significant effect of the related glycoprotein transferrin [54]. Some biomarkers that
have been implicated in other studies–such as soluble CD14 [16,17], endotoxin core antibodies
(EndoCAB) [12], zonulin, intestinal fatty acid binding protein [35], retinol binding protein
and calprotectin [17]–were not included in any of the panels. Others were excluded from the
analysis due to having too few unique observations, notably almost all the interleukins, which
were only tested for in the case-control panel, a limitation of this study.
Several other limitations warrant highlighting. Most associations that were apparently sta-
tistically significant in the single biomarker models appeared much less so after accounting for
the FDR–indeed, only adiponectin remained significant at the Bonferroni-corrected α level.
Furthermore, the results of the adjusted subset models do not account for the variable selection
in the first stage SCAD model, a post-selection inference problem that can lead to inflated
type-1 errors and overly narrow confidence intervals [55]. However, the associations identified
by this analysis should be assessed, not just by their statistical significance but by their

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 15 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

biological plausibility and in light of the fact that the biomarkers selected for the subset and the
relative strength of their associations with the outcome are broadly consistent with known bio-
logical pathways. Another limitation is the assumption both in the subset selection stage and
in fitting the final models that any relationships between biomarkers and LAZ-score would be
linear. Exploratory analysis revealed some evidence to challenge this, which may limit the
accuracy of the predictions from the linear models, however further analysis using multivariate
regression splines suggested that only a very small number of biomarkers were affected by this
assumption. As consensus develops around a final set of important biomarkers of EED such
non-linear effects will need to be more rigorously characterized.
Applying the penalized regression models to the database that included the observations at
24 months of age, did not improve the predictive capability of the model. Similarly, the final 7-
24-month model explained a smaller proportion of the variance in the outcome than the 7-
15-month model. However, for some biomarkers that were included in both models, the 7-
24-month model tended to give larger and more statistically significant effect size estimates
than the 7-15-month model (with the notable exception of hemoglobin). The reason for the
difference in explanatory power may be because the 7-24-month database did not include
transferrin (which was not tested at 24 months of age), the biomarker with the largest effect
size in the 7-15-month final model.
Studies with more intensive sample collection and frequent follow-up are needed to explore
random effects and short-term intra- and inter-subject variability of these biomarkers as well
as those that were excluded from this analysis and to more precisely model their effects on
growth [11]. The validity of these biomarkers as clinically relevant predictors of growth in new
populations can be readily assessed given that ELISA kits for most of them are commercially
available. This is important considering the high burden of stunting in under-resourced set-
tings in low- and middle-income countries where these biomarkers can potentially be tested in
regional laboratories, and the results used to inform care and programs aimed at controlling
stunting.
The expanded testing of analytes chosen for their characterization as being important
immune and metabolic regulators pertinent to child growth revealed several important find-
ings. This selected subset of biomarkers explained 17.7–23.0% of the variance in LAZ score
with measurements taken at 2 or 3 time points, compared to a single biomarker such as MPO
which only accounted for 2.8% of the variance with monthly follow-up up to age 3 years in the
same population [11]. Future studies should aim to characterize changes in LAZ scores when
assessing the interaction between EED biomarkers and intestinal infections by specific patho-
gens. These plasma biomarkers represent a set of surrogate outcomes which can be measured
at different time points, all of which are characteristic of a good biomarker of EED to circum-
vent the problems associated with the lactulose/mannitol test, the current gold standard test
(such as the variable in its association with child growth, which, even when significant has an
effect size that is much smaller than the selected panel described here) [56].
In summary, penalized regression modeling approaches–most notably SCAD—can be used
to select subsets from large panels of candidate biomarkers of EED providing translational
value in the form of further evidence for known markers and in generating hypotheses about
new ones. Adiponectin, IL-8, proline, SAP and transferrin, among others, are promising
plasma biomarkers of EED.

Supporting information
S1 List. Biomarkers quantified in each panel and their units.
(PDF)

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 16 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

S1 Table. Summary statistics of candidate biomarkers.

(PDF)
S2 Table. Coefficient estimates (with 95% confidence intervals) from linear regression
models for biomarkers selected by SCAD along with the predicted difference in child’s
height 2 months after the last sample for children at the 25th and 75th percentile of the bio-
marker distribution adjusted for contemporaneous LAZ-score.
(PDF)
S3 Table. STROBE checklist.
(PDF)
S1 Fig. Participant flowchart.
(TIF)
S2 Fig. Polynomial smooth plot of the relationship between plasma Eotaxin-3 concentra-
tion and lagged LAZ-scores.
(TIF)
S3 Fig. Polynomial smooth plot of the relationship between plasma citrulline concentra-
tion and lagged LAZ-scores.
(TIF)
S4 Fig. Polynomial smooth plot of the relationship between plasma myoglobin concentra-
tion and lagged LAZ-scores.
(TIF)
S5 Fig. Polynomial smooth plot of the relationship between urinary lactulose concentra-
tion and lagged LAZ-scores.
(TIF)
S6 Fig. Polynomial smooth plot of the relationship between plasma Sex Hormone-Binding
Globulin concentration and lagged LAZ-scores.
(TIF)

Acknowledgments
We wish to thank participants, their families and the study community for their dedicated
time and effort to better the understanding the transmission and more enduring impact of
enteric infections in early childhood. We would also like to thank Drs. Leah Jager (JHSPH)
and William Pan (Duke University) for consultation regarding the statistical analysis, and Dr.
Ben Jann (University of Bern, Switzerland) for guidance in generating the figures. We would
like to acknowledge support for the statistical analysis from the National Center for Research
Resources and the National Center for Advancing Translational Sciences (NCATS) of the
National Institutes of Health through Grant Number 1UL1TR001079.

Author Contributions
Conceptualization: Margaret N. Kosek.
Data curation: Josh M. Colston.
Formal analysis: Josh M. Colston, Lawrence H. Moulton.
Funding acquisition: Margaret N. Kosek.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 17 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

Investigation: Pablo Peñataro Yori, Maribel Paredes Olortegui, Peter S. Kosek, Dixner Rengifo
Trigoso, Mery Siguas Salas, Fahmina Fardus-Reid, Jonathan R. Swann, Margaret N. Kosek.
Methodology: Margaret N. Kosek.
Writing – original draft: Josh M. Colston.
Writing – review & editing: Francesca Schiaffino, Ruthly François, Margaret N. Kosek.

References
1. Black RE, Allen LH, Bhutta ZA, Caulfield LE, de Onis M, Ezzati M, et al. Maternal and child undernutri-
tion: global and regional exposures and health consequences. Lancet (London, England). 2008; 371:
243–60. https://doi.org/10.1016/S0140-6736(07)61690-0
2. Victora CG, de Onis M, Hallal PC, Blössner M, Shrimpton R. Worldwide timing of growth faltering: revis-
iting implications for interventions. Pediatrics. 2010; 125: e473–80. https://doi.org/10.1542/peds.2009-
1519 PMID: 20156903
3. Dewey KG, Adu-Afarwuah S. Systematic review of the efficacy and effectiveness of complementary
feeding interventions in developing countries. Matern Child Nutr. 2008; 4: 24–85. https://doi.org/10.
1111/j.1740-8709.2007.00124.x PMID: 18289157
4. Harper KM, Mutasa M, Prendergast AJ, Humphrey J, Manges AR. Environmental enteric dysfunction
pathways and child stunting: A systematic review. PLoS Negl Trop Dis. 2018; 12. https://doi.org/10.
1371/journal.pntd.0006205 PMID: 29351288
5. Arndt MB, Walson JL. Enteric infection and dysfunction—A new target for PLOS Neglected Tropical
Diseases. Ryan ET, editor. PLoS Negl Trop Dis. 2018; 12: e0006906. https://doi.org/10.1371/journal.
pntd.0006906 PMID: 30592716
6. Kelly P, Menzies I, Crane R, Zulu I, Nickols C, Feakins R, et al. Responses of small intestinal architec-
ture and function over time to environmental factors in a tropical population. Am J Trop Med Hyg. 2004;
70: 412–9. Available: http://www.ncbi.nlm.nih.gov/pubmed/15100456 PMID: 15100456
7. Korpe PS, Petri WA. Environmental enteropathy: critical implications of a poorly understood condition.
Trends Mol Med. 2012; 18: 328–36. https://doi.org/10.1016/j.molmed.2012.04.007 PMID: 22633998
8. Kosek MN, Ahmed T, Bhutta Z, Caulfield L, Guerrant R, Houpt E, et al. Causal Pathways from Entero-
pathogens to Environmental Enteropathy: Findings from the MAL-ED Birth Cohort Study. EBioMedi-
cine. 2017; 18: 109–117. https://doi.org/10.1016/j.ebiom.2017.02.024 PMID: 28396264
9. Kosek M, Haque R, Lima A, Babji S, Shrestha S, Qureshi S, et al. Fecal Markers of Intestinal Inflamma-
tion and Permeability Associated with the Subsequent Acquisition of Linear Growth Deficits in Infants.
Am J Trop Med Hyg. 2013; 88: 390–396. https://doi.org/10.4269/ajtmh.2012.12-0549 PMID: 23185075
10. Keusch GT, Rosenberg IH, Denno DM, Duggan C, Guerrant RL, Lavery J V., et al. Implications of
Acquired Environmental Enteric Dysfunction for Growth and Stunting in Infants and Children Living in
Low- and Middle-Income Countries. Food Nutr Bull. 2013; 34: 357–364. https://doi.org/10.1177/
156482651303400308 PMID: 24167916
11. Colston JM, Peñataro Yori P, Colantuoni E, Moulton LH, Ambikapathi R, Lee G, et al. A methodologic
framework for modeling and assessing biomarkers of environmental enteropathy as predictors of
growth in infants: an example from a Peruvian birth cohort. Am J Clin Nutr. 2017; 106: 245–55. https://
doi.org/10.3945/ajcn.116.151886 PMID: 28592604
12. Hoke MK, McCabe KA, Miller AA, McDade TW. Validation of endotoxin-core antibodies in dried blood
spots as a measure of environmental enteropathy and intestinal permeability. Am J Hum Biol. 2018;
e23120. https://doi.org/10.1002/ajhb.23120 PMID: 29532544
13. Faubion WA, Camilleri M, Murray JA, Kelly P, Amadi B, Kosek MN, et al. Improving the detection of
environmental enteric dysfunction: a lactulose, rhamnose assay of intestinal permeability in children
aged under 5 years exposed to poor sanitation and hygiene. BMJ Glob Heal. 2016; 1: e000066. https://
doi.org/10.1136/bmjgh-2016-000066 PMID: 28588929
14. Kosek MN, Mduma E, Kosek PS, Lee GO, Svensen E, Pan WKY, et al. Plasma Tryptophan and the
Kynurenine-Tryptophan Ratio are Associated with the Acquisition of Statural Growth Deficits and Oral
Vaccine Underperformance in Populations with Environmental Enteropathy. Am J Trop Med Hyg. 2016;
95: 928–937. https://doi.org/10.4269/ajtmh.16-0037 PMID: 27503512
15. Breen EC, Reynolds SM, Cox C, Jacobson LP, Magpantay L, Mulder CB, et al. Multisite comparison of
high-sensitivity multiplex cytokine assays. Clin Vaccine Immunol. 2011; 18: 1229–42. https://doi.org/10.
1128/CVI.05032-11 PMID: 21697338

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 18 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

16. Lu M, Zhou J, Naylor C, Kirkpatrick BD, Haque R, Petri WA, et al. Application of penalized linear regres-
sion methods to the selection of environmental enteropathy biomarkers. Biomark Res. 2017; 5: 9.
https://doi.org/10.1186/s40364-017-0089-4 PMID: 28293424
17. Naylor C, Lu M, Haque R, Mondal D, Buonomo E, Nayak U, et al. Environmental Enteropathy, Oral Vac-
cine Failure and Growth Faltering in Infants in Bangladesh. EBioMedicine. 2015; 2: 1759–66. https://
doi.org/10.1016/j.ebiom.2015.09.036 PMID: 26870801
18. Moreau GB, Ramakrishnan G, Cook HL, Fox TE, Nayak U, Ma JZ, et al. Childhood growth and neuro-
cognition are associated with distinct sets of metabolites. EBioMedicine. 2019; 44: 597–606. https://doi.
org/10.1016/j.ebiom.2019.05.043 PMID: 31133540
19. Yori PP, Lee G, Olortegui MP, Chavez CB, Flores JT, Vasquez AO, et al. Santa Clara de Nanay: The
MAL-ED Cohort in Peru. Clin Infect Dis. 2014; 59: S310–S316. https://doi.org/10.1093/cid/ciu460
PMID: 25305303
20. MAL-ED Network Investigators The MAL-ED Network Investigators, MAL-ED Network Investigators.
The MAL-ED study: a multinational and multidisciplinary approach to understand the relationship
between enteric pathogens, malnutrition, gut physiology, physical growth, cognitive development, and
immune responses in infants and children up to 2 years of. Clin Infect Dis. 2014; 59 Suppl 4: S193–206.
https://doi.org/10.1093/cid/ciu653 PMID: 25305287
21. WHO Multicentre Growth Reference Study Group. WHO Child Growth Standards: Length/height-for-
age, weight-for-age, weight-for-length, weight-for-height and body mass index-for-age: Methods and
development. Geneva: World Health Organization; 2006. Available: http://www.who.int/childgrowth/
standards/technical_report/en/
22. Kosek M, Guerrant RL, Kang G, Bhutta Z, Yori PP, Gratz J, et al. Assessment of environmental enterop-
athy in the MAL-ED cohort study: theoretical and analytic framework. Clin Infect Dis. 2014; 59 Suppl 4:
S239–47. https://doi.org/10.1093/cid/ciu457 PMID: 25305293
23. Richard SA, McCormick BJJ, Miller MA, Caulfield LE, Checkley W. Modeling Environmental Influences
on Child Growth in the MAL-ED Cohort Study: Opportunities and Challenges. Clin Infect Dis. 2014; 59:
S255–S260. https://doi.org/10.1093/cid/ciu436 PMID: 25305295
24. Myriad RBM. HumanMAP v. 2.0. 2018 [cited 22 Aug 2018]. Available: https://myriadrbm.com/products-
services/humanmap-services/humanmap/
25. Gray N, Zia R, King A, Patel VC, Wendon J, McPhail MJW, et al. High-Speed Quantitative UPLC-MS
Analysis of Multiple Amines in Human Plasma and Serum via Precolumn Derivatization with 6-Amino-
quinolyl- N -hydroxysuccinimidyl Carbamate: Application to Acetaminophen-Induced Liver Failure. Anal
Chem. 2017; 89: 2478–2487. https://doi.org/10.1021/acs.analchem.6b04623 PMID: 28194962
26. McCormick BJJ, Lee GO, Seidman JC, Haque R, Mondal D, Quetz J, et al. Dynamics and Trends in
Fecal Biomarkers of Gut Function in Children from 1–24 Months in the MAL-ED Study. Am J Trop Med
Hyg. 96. https://doi.org/10.4269/ajtmh.16-0496 PMID: 27994110
27. Ahmed SF, Tucker P, Mushtaq T, Wallace AM, Williams DM, Hughes IA. Short-term effects on linear
growth and bone turnover in children randomized to receive prednisolone or dexamethasone. Clin
Endocrinol (Oxf). 2002; 57: 185–191. https://doi.org/10.1046/j.1365-2265.2002.01580.x PMID:
12153596
28. Bath LE, Crofton PM, Evans AEM, Ranke MB, Elmlinger MW, Kelnar CJH, et al. Bone Turnover and
Growth during and after Chemotherapy in Children with Solid Tumors. Pediatr Res. 2004; 55: 224–230.
https://doi.org/10.1203/01.PDR.0000100903.83472.09 PMID: 14605245
29. Isanaka S, Kodish SR, Berthé F, Alley I, Nackers F, Hanson KE, et al. Outpatient treatment of severe
acute malnutrition: Response to treatment with a reduced schedule of therapeutic food distribution. Am
J Clin Nutr. 2017; 105: 1191–1197. https://doi.org/10.3945/ajcn.116.148064 PMID: 28404577
30. Hornung RW, Reed LD. Estimation of Average Concentration in the Presence of Nondetectable Values.
Appl Occup Environ Hyg. 1990; 5: 46–51. https://doi.org/10.1080/1047322X.1990.10389587
31. Schafer JL (Joseph L. Analysis of incomplete multivariate data. Chapman & Hall; 1997. Available:
https://www.crcpress.com/Analysis-of-Incomplete-Multivariate-Data/Schafer/p/book/9780412040610
32. Storey JD, Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad Sci U S A.
2003; 100: 9440–9445. https://doi.org/10.1073/pnas.1530509100 PMID: 12883005
33. Simes RJ. An improved bonferroni procedure for multiple tests of significance. Biometrika. 1986; 73:
751–754. https://doi.org/10.1093/biomet/73.3.751
34. StataCorp. Stata Statistical Software: Release 15. College Station, TX; 2017.
35. Guerrant RL, Leite AM, Pinkerton R, Medeiros PHQS, Cavalcante PA, DeBoer M, et al. Biomarkers of
Environmental Enteropathy, Inflammation, Stunting, and Impaired Growth in Children in Northeast Bra-
zil. PLoS One. 2016; 11: e0158772. https://doi.org/10.1371/journal.pone.0158772 PMID: 27690129

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 19 / 20

 Biomarkers of environmental enteric dysfunction and nutritional status

36. Soliman AT, De Sanctis V, Kalra S. Anemia and growth. Indian J Endocrinol Metab. 2014; 18: S1–5.
https://doi.org/10.4103/2230-8210.145038 PMID: 25538873
37. Richard SA, Mccormick BJJ, Murray-Kolb LE, Lee GO, Seidman JC, Mahfuz M, et al. Enteric dysfunc-
tion and other factors associated with attained size at 5 years: MAL-ED birth cohort study findings. Am J
Clin Nutr. 2019; 110: 131–138. https://doi.org/10.1093/ajcn/nqz004 PMID: 31127812
38. Hossain M, Nahar B, Haque MA, Mondal D, Mahfuz M, Naila NN, et al. Serum Adipokines, Growth Fac-
tors, and Cytokines Are Independently Associated with Stunting in Bangladeshi Children. Nutrients.
2019; 11. https://doi.org/10.3390/nu11081827 PMID: 31394828
39. Kamng’ona AW, Young R, Arnold CD, Kortekangas E, Patson N, Jorgensen JM, et al. The association
of gut microbiota characteristics in Malawian infants with growth and inflammation. Sci Rep. 2019; 9:
12893. https://doi.org/10.1038/s41598-019-49274-y PMID: 31501455
40. Bharadwaj S, Ginoya S, Tandon P, Gohel TD, Guirguis J, Vallabh H, et al. Malnutrition: laboratory mark-
ers vs nutritional assessment. Gastroenterol Rep. 2016; 4: 272–280. https://doi.org/10.1093/gastro/
gow013 PMID: 27174435
41. Holst JJ. The Physiology of Glucagon-like Peptide 1. Physiol Rev. 2007; 87: 1409–1439. https://doi.org/
10.1152/physrev.00034.2006 PMID: 17928588
42. Yeung EH, Sundaram R, Xie Y, Lawrence DA. Newborn adipokines and early childhood growth. Pediatr
Obes. 2018; 13: 505–513. https://doi.org/10.1111/ijpo.12283 PMID: 29781193
43. Woo JG, Guerrero ML, Altaye M, Ruiz-Palacios GM, Martin LJ, Dubert-Ferrandon A, et al. Human milk
adiponectin is associated with infant growth in two independent cohorts. Breastfeed Med. 2009; 4: 101–
9. https://doi.org/10.1089/bfm.2008.0137 PMID: 19500050
44. Semba RD, Shardell M, Sakr Ashour FA, Moaddel R, Trehan I, Maleta KM, et al. Child Stunting is Asso-
ciated with Low Circulating Essential Amino Acids. EBioMedicine. 2016; 6: 246–252. https://doi.org/10.
1016/j.ebiom.2016.02.030 PMID: 27211567
45. Wang F-M, Lin C-M, Lien S-H, Wu L-W, Huang C-F, Chu D-M. Sex difference determined the role of
sex hormone-binding globulin in obese children during short-term weight reduction program. Medicine
(Baltimore). 2017; 96: e6834. https://doi.org/10.1097/MD.0000000000006834 PMID: 28489766
46. Hutchinson WL, Hohenester E, Pepys MB. Human serum amyloid P component is a single uncom-
plexed pentamer in whole serum. Mol Med. 2000; 6: 482–93. Available: http://www.ncbi.nlm.nih.gov/
pubmed/10972085 PMID: 10972085
47. Agrawal A, Singh PP, Bottazzi B, Garlanda C, Mantovani A. Pattern recognition by pentraxins. Adv Exp
Med Biol. 2009; 653: 98–116. Available: http://www.ncbi.nlm.nih.gov/pubmed/19799114 https://doi.org/
10.1007/978-1-4419-0901-5_7 PMID: 19799114
48. Poulsen ET, Pedersen KW, Marzeda AM, Enghild JJ. Serum Amyloid P Component (SAP) Interactome
in Human Plasma Containing Physiological Calcium Levels. Biochemistry. 2017; 56: 896–902. https://
doi.org/10.1021/acs.biochem.6b01027 PMID: 28098450
49. Tortora GJ, Derrickson BH. Principles of Anatomy and Physiology. 14th ed. Wiley; 2014. Available:
http://www.wiley.com/WileyCDA/WileyTitle/productCd-EHEP002935.html
50. Kim H-Y, Mohan S. Role and Mechanisms of Actions of Thyroid Hormone on the Skeletal Development.
Bone Res. 2013; 1: 146–161. https://doi.org/10.4248/BR201302004 PMID: 26273499
51. Iqbal NT, Sadiq K, Syed S, Akhund T, Umrani F, Ahmed S, et al. Promising Biomarkers of Environmen-
tal Enteric Dysfunction: A Prospective Cohort study in Pakistani Children. Sci Rep. 2018; 8: 2966.
https://doi.org/10.1038/s41598-018-21319-8 PMID: 29445110
52. Dabrowska AM, Tarach JS, Wojtysiak-Duma B, Duma D. Fetuin-A (AHSG) and its usefulness in clinical
practice. Review of the literature. Biomed Pap. 2015; 159: 352–359. https://doi.org/10.5507/bp.2015.
018 PMID: 25916279
53. Kosek MN, Lee GO, Guerrant RL, Haque R, Kang G, Ahmed T, et al. Age and Sex Normalization of
Intestinal Permeability Measures for the Improved Assessment of Enteropathy in Infancy and Early
Childhood. J Pediatr Gastroenterol Nutr. 2017; 65: 31–39. https://doi.org/10.1097/MPG.
0000000000001610 PMID: 28644347
54. Ponka P, Beaumont C, Richardson DR. Function and regulation of transferrin and ferritin. Semin Hema-
tol. 1998; 35: 35–54. Available: http://www.ncbi.nlm.nih.gov/pubmed/9460808 PMID: 9460808
55. Taylor J, Tibshirani RJ. Statistical learning and selective inference. Proc Natl Acad Sci U S A. 2015;
112: 7629–7634. https://doi.org/10.1073/pnas.1507583112 PMID: 26100887
56. Denno DM, VanBuskirk K, Nelson ZC, Musser CA, Hay Burgess DC, Tarr PI. Use of the lactulose to
mannitol ratio to evaluate childhood environmental enteric dysfunction: A systematic review. Clin Infect
Dis. 2014. https://doi.org/10.1093/cid/ciu541 PMID: 25305289

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007851 November 15, 2019 20 / 20

View publication stats