Espira_2023

PLOS NEGLECTED TROPICAL DISEASES
RESEARCH ARTICLE
Stool biomarkers as measures of enteric

pathogen infection in infants from Addis
Ababa informal settlements
Leon M. Espira ID1, Gwenyth O. Lee1,2, Kaleab Baye3, Andrew D. Jones4, Nancy G. Love5,
Joseph N. S. Eisenberg1*
1 Department of Epidemiology, University of Michigan School of Public Health, Ann Arbor, Michigan, United
States of America, 2 Rutgers Global Health Institute & Department of Biostatistics and Epidemiology School
of Public Health Rutgers, The State University of New Jersey, New Brunswick, New Jersey, United States of
America, 3 Center for Food Science and Nutrition, College of Natural and Computational Sciences, Addis
Ababa University, Addis Ababa, Ethiopia, 4 Department of Nutritional Sciences, University of Michigan
School of Public Health, Ann Arbor, Michigan, United States of America, 5 Department of Civil and
Environmental Engineering, University of Michigan, Ann Arbor, Michigan, United States of America
a1111111111
a1111111111 * jnse@umich.edu
a1111111111
a1111111111
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Abstract
Frequent enteric infections in children may be an important cause of growth faltering; how-
ever, we do not fully understand the mechanisms by which pathogen infections and the
OPEN ACCESS physiological responses to these infections result in poorer growth. Commonly used protein
Citation: Espira LM, Lee GO, Baye K, Jones AD, fecal biomarkers (anti-alpha trypsin, neopterin, and myeloperoxidase) provide broad immu-
Love NG, Eisenberg JNS (2023) Stool biomarkers nological information on an inflammatory response; however, they do not provide informa-
as measures of enteric pathogen infection in
tion on non-immune processes (e.g., gut integrity) that may be important indicators of
infants from Addis Ababa informal settlements.
PLoS Negl Trop Dis 17(2): e0011112. https://doi. chronic end states such as environmental enteric dysfunction (EED). To explore how addi-
org/10.1371/journal.pntd.0011112 tional biomarkers will better inform which physiological pathways (both immune and non-
Editor: James Michael Fleckenstein, Washington immune) are impacted by pathogen exposure we added to the traditional panel of 3 protein
University School of Medicine, UNITED STATES fecal biomarkers 4 novel fecal mRNA transcript biomarkers (sucrase isomaltase, caudal
Received: July 18, 2022 homeobox 1, S100A8, and mucin 12) and analyzed stool samples from infants living in infor-
mal settlements in Addis Ababa, Ethiopia. To test how this expanded biomarker panel cap-
Accepted: January 19, 2023
tures distinct pathogen exposure processes we used two different scoring systems. First,
Published: February 21, 2023
we used a theory-based approach to assign each biomarker to specific physiological attri-
Copyright: © 2023 Espira et al. This is an open butes based on prior understanding of each biomarker. Second, we used data reduction
access article distributed under the terms of the
methods to categorize biomarkers and then assign physiological attributes to those catego-
Creative Commons Attribution License, which
permits unrestricted use, distribution, and ries. We used linear models to examine the association between the derived biomarker
reproduction in any medium, provided the original scores (based on mRNA and protein levels) and stool pathogen gene counts to determine
author and source are credited. pathogen specific effects on gut physiology and immune responses. Inflammation scores
Data Availability Statement: The data is available were positively associated with Shigella and enteropathogenic E.Coli (EPEC) infection,
from Dryad repository DOI:10.5061/dryad. while gut integrity scores were negatively associated with Shigella, EPEC and, shigatoxi-
7pvmcvdwq.
genic E.coli (STEC) infection. Our expanded panel of biomarkers hold promise as tools to
Funding: The authors received no specific funding measure systemic outcomes of enteric pathogen infection. mRNA biomarkers complement
for this work.
established protein biomarkers by providing important cell-specific physiological and
Competing interests: The authors have no
competing interests to disclose.
PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 1 / 18

PLOS NEGLECTED TROPICAL DISEASES Stool biomarkers as measures of enteric pathogen infection in infants
immunological consequences of pathogen carriage that can lead to chronic end states such
as EED.
Author summary
Frequent enteric infections in children may be an important cause of growth faltering.
Mechanisms by which pathogen infections result in poorer growth, however, are not fully
understood. Commonly used biomarkers provide information on broad immunological
processes but not on non-immune processes (e.g., gut integrity) that may be important
indicators of overall health. In this manuscript we explore how additional biomarkers can
expand our understanding on which immune and non-immune pathways are impacted
by pathogen exposure. To do this we developed a panel consisting of 3 commonly used
and 4 novel biomarkers. We analyzed stool samples from infants living in informal settle-
ments in Addis Ababa, Ethiopia. To test how this expanded biomarker panel captures dis-
tinct pathological processes we used two scoring systems and examined their association
with stool pathogen gene counts. We found that Shigella and enteropathogenic E. Coli
(EPEC) were positively associated in inflammation, while the integrity of intestinal tissue
was negatively associated with Shigella, EPEC and shigatoxigenic E.coli (STEC) infection.
Our biomarker panel holds promise as a tool to measure both immune and non-immune
outcomes of pathogen infection.
Introduction
Infant growth faltering remains a persistent concern in low- and middle-income countries
globally, where approximately 165 million children under 5 years of age are stunted [1]. It has
long been recognized that enteric infections are an important cause of growth faltering; how-
ever, the mechanisms by which frequent pathogen infections and the physiological responses
to these infections result in poorer growth remain poorly understood. One putative mechanis-
tic linking enteric pathogen infection and growth faltering is a condition known as environ-
mental enteric dysfunction (EED).
EED results in altered small bowel physiology due to repeated enteric infections. EED has
demonstrable histological features such as villous flattening, crypt hyperplasia and lympho-
cytic infiltration of the lamina propria coupled with chronic intestinal inflammation [2–6],
which have been associated with long-term growth faltering and stunting. EED can compro-
mise the integrity of the gut and allow potentially pathogenic bacteria to cross the gut wall,
triggering a chronic inflammatory process [7]. Inflammation may directly down-regulate
growth, as well as indirectly affect growth through appetite suppression [7,8]. Because EED
may compromise the integrity of the intestinal wall, an abnormally large number of white
blood cells can infiltrate the gut, forcing an infant’s metabolism to manage a chronically
stressed immune system [7,9,10].
Diagnosing EED has been a challenge, potentially because EED is likely not a single syn-
drome, but the result of multiple contributing processes that may differ geographically [11].
An effective diagnostic tool, therefore, is one that captures specific, critical aspects of gut
health, such as enterocyte damage. Asymptomatic pathogen carriage is common among young
children living without access to improved water and sanitation. This potentially hinders the
use of diagnostic techniques that rely on the measurement of immunological responses, since

asymptomatic pathogen carriage often results in transient immune activation [12,13]. Asymp-
tomatic pathogen carriage still causes cellular and tissue damage, reinforcing the need for diag-
nostic tools that provide cell and tissue specific measures of immune state and tissue integrity.
The gold standard for diagnosis of EED is the histological examination of intestinal tissue.
However, because of the invasive nature of collecting biopsies from very young children, there
have been concerted efforts to develop alternative non-invasive tests. The most common non-
invasive test is the lactulose: mannitol (L:M) urine test. The L:M test is designed to measure
intestinal permeability but provides no information on the immunological state of subjects
[9,10]. The L:M test is also challenging to administer and results are hard to compare across
dosing and urine collection protocols and analytic platforms [14].
Another frequently reported EED measure is a biomarker panel consisting of three proteins
isolated from stool: alpha-1-antitrypsin (AAT), neopterin, and myeloperoxidase (MPO) [15].
Human alpha-1 antitrypsin is a water soluble glycoprotein [16]. During intestinal inflamma-
tion, AAT is extraverted into the gut and is therefore a marker of intestinal permeability and
protein loss [15]. Neopterin is synthesized primarily by activated monocytes, macrophages,
dendritic cells, and endothelial cells [17]. The production of neopterin by macrophages is a
marker of INF-γ mediated activation of macrophages by TH1 cells, indicative of an adaptive
immune response; i.e., the pathogens driving this response have previously been encountered
[18,19]. Neopterin has been used to diagnose autoimmune diseases such as celiac disease,
which is considered a clinical and histopathological analog of EED [15]. MPO is a marker of
the neutrophil response and has been correlated with disease activity in inflammatory bowel
disease [15].
These three protein stool biomarkers are easier to measure than the lactulose: mannitol
ratio and provide greater immunological detail and information about permeability. To
improve specificity even more, mRNA transcripts specific for intestinal inflammation have
recently been investigated [20–23]. Several features make these transcripts promising for the
assessment of EED: 1) they are able to target and measure a range of intestinal processes
related to the immune system much like AAT, neopterin, and MPO; 2) they are able to mea-
sure other non-immune processes such as epithelial state and nutrient absorption that are
important indicators of EED; and 3) their measurement can be multiplexed, allowing for mul-
tiple markers to be measured simultaneously. Because mRNA transcript biomarkers can be
cell specific, they may be particularly valuable in providing cellular level information on spe-
cific physiological and immunological processes.
These mRNA transcripts have been described and measured in populations of rural infants
and children in Malawi and Sierra Leone, where they showed promise as indicators of EED
based on their association with L:M test results and inflammatory indicators [20–25]. How-
ever, several questions remain about the use of these biomarkers for the assessment of EED. Of
particular interest is whether these biomarkers accurately measure the features of EED (i.e.,
gut integrity and immunological activation) that are critical to child health.
In this manuscript, we examine the degree to which our expanded biomarker panel can dis-
tinguish among pathogens; specifically, we aim to distinguish between those that are enteroin-
vasive compared to those that attach to enterocyte surfaces, and those that form biofilms. We
do this by analyzing stool samples from infants living in informal settlements in Addis Ababa,
Ethiopia.
Our biomarker panel comprises three established fecal protein biomarkers (AAT, neop-
terin, and MPO) and 4 novel mRNA transcript biomarkers (sucrase isomaltase (SI), caudal
homeobox 1 (Cdx1), S100A8 and mucin 12). SI and Cdx1 were selected to inform non-inflam-
matory processes, those related to gut integrity. SI is located on the brush border membrane of
enterocytes, and SI levels are reduced with mucosal injury [26]. Caudal homeobox proteins are

global transcription factors, and a close homologue of Cdx1 has been shown to transactivate SI
[27]. Given the similarities, we argue that Cdx1 can be used as a marker of gut integrity along
with SI. S100A8, which is part of the heterodimeric protein S100A8/A9 (calprotectin) [28],
was selected to mirror the neutrophil response measured by MPO. Finally, Mucin 12 is a mem-
brane bound mucin constitutively expressed by enterocytes mainly in the colon, and is there-
fore a third potential marker of gut integrity [29,30].
Materials and methods

Ethical approval
Ethical approval for sample collection and surveys was granted by institutional review boards
at the University of Michigan (HUM00115103), and the Addis Ababa University (IRB/029/
2017). Parents or legal guardians gave verbal, informed consent prior to participation or col-
lection of data. Fecal samples were transported with clearance obtained from the Food, Medi-
cine, and Health Care Administration, and Control Authority in Ethiopia.
Study population
Stool samples were collected in 2018 from 136 infants aged 6–23 months across 12 localities
in Addis Ababa, Ethiopia. Based on available resources, our goal was to obtain 8–12 stool
samples per locality (across all 12 localities enrolled in our larger water and sanitation
study). Within each locality, health extension workers identified all households with infants
aged 6–23 months and compiled them into a list. In the larger study, every second or third
household was systematically selected (depending on the number of households in the local-
ity). For this biomarker study, 12 households from each locality were randomly selected
from the large study sample and asked to provide stool samples. A key informant was asked
to respond to a survey on sanitation access and consent to the collection of anthropometric
data.
Stool sample processing

After collection, stool samples were catalogued and scored for consistency (formed, soft or
watery). Samples were stored at 4˚C overnight before processing the following morning. Ali-
quots of the stool samples, to be used for ELISA biomarker measurements, was stored at
-80˚C. Aliquots were also processed for nucleic acid extraction using the ZymoBiomics DNA/
RNA Mini Kit (Zymo Research, Irvine, CA).
ELISA protein biomarker measurements

ELISA’s for three protein biomarkers (AAT, neopterin, and MPO) were run on-site at the
Ethiopian Institute of Public Health as per the manufacturer’s instructions. AAT levels were
determined using the Human Alpha-1-Antitrypsin ELISA from Biovendor Research and Diag-
nostic Products (Brno, Czech Republic) kit. Neopterin levels were determined using the Gen-
Way Biotech Inc. (San Diego, USA) Neopterin ELISA. MPO levels were measured using the
IDK MPO ELISA from Immunodiagnostik AG (Bensheim, Germany). Prior to running the
ELISA assays, stool aliquots were thawed on ice and thereafter stored at 4˚C until the assays
were run.
Nucleic acid extraction

Standard ZymoBiomics DNA/RNA Mini Kit (Zymo Research, Irvine, CA) protocol for nucleic
acid isolation from fecal samples was used. Briefly, 200mg of stool was weighed out and placed

in screw cap microcentrifuge tubes containing the DNA/RNA Shield Lysis Buffer. The tubes
were packed in leak proof containers for transport to the University of Michigan where DNA
and RNA were extracted as specified in the manufacturer’s protocol within two months of
sample collection. The quality and concentration of the extracted nucleic acids was measured
using a Nanodrop Spectrophotometer (Thermo Scientific, Waltham, MA). The DNA and
RNA were used to detect mRNA transcripts and pathogens.
Droplet digital PCR detection of mRNA transcripts

Sample mRNA transcript counts were quantified using the QX200 Droplet Digital PCR
(ddPCR) system (Bio-Rad, Hercules, CA) using duplexed FAM and VIC TaqMan assays.
Assay setup and cycling conditions are provided in the S1 Text.
Droplet digital PCR quantification of stool pathogen gene counts

Pathogen gene counts were quantified using the QX200 Droplet Digital PCR system (Bio-Rad,
Hercules, CA). Assay setup and cycling conditions are provided in the S2 Text. Stool samples
were screened for six bacteria (enteroaggregative E.coli (EAEC), enteropathogenic E.coli
(EPEC), enterotoxigenic E.coli (ETEC), shigatoxigenic E.coli (STEC), Shigella, and Campylo-
bacter spp), two protozoans (Giardia lamblia, and Cryptosporidium spp), and one virus (noro-
virus GI and GII). EPEC was screened for two gene targets (S1 Table).
Droplet digital PCR data processing

All sample quantification was carried out using QuantaSoft software (Bio-Rad, Hercules, CA).
Wells were checked and samples with <10,000 accepted droplets were rerun. To check the
inter assay variability, 1/3 of the samples on a 96-well plate were randomly selected to be re-
run for both mRNA transcript and pathogen quantification, and coefficients of variation (CV)
were calculated. If more than 5% of re-run samples had CVs higher than 15%, the entire
96-well plate from which the samples were selected from was re-run.
The quantification of mRNA transcripts was carried out by visually setting thresholds to
distinguish between negative and positive droplets [20,21]. The concentration in each well was
then normalized to GAPDH and results presented as Target/GAPDH ratios.
To quantify stool pathogen gene counts, the threshold for differentiating negative from pos-
itive droplets was defined as one standard deviation above the negative droplet threshold on
the no template controls [31,32]. Following the setting of the threshold, all wells were visually
inspected. Wells with less than three positive droplets were considered negative and assigned a
value equal to 0.1 prior to the calculation of final pathogen gene concentrations. This avoids
the log transformation of zero values [31]. Final concentrations of gene copies per 200mg of
stool were obtained by multiplying by appropriate dilution factors. Final concentrations were
then converted to log10.
The data from the ELISA and ddPCR analysis is deposited in Dryad under the unique digi-
tal object identifier (DOI): doi:10.5061/dryad.7pvmcvdwq [33].
Development of Physiological Scores and Statistical Analysis

We develop two summary scores based on our panel of 3 protein and 4 mRNA biomarkers: 1)
“theory driven” scores similar to that described by Liu et al. (2020) (S2 Table) [11] and 2) “data
derived” PCA scores (Fig 1).
The “theory driven” scores were derived by sorting the biomarkers into quintiles with the
lowest quintile assigned a grade score of 0 and the highest quintile assigned a grade score of 4

Fig 1. Derivation of the two scoring systems for the analysis. The protein biomarkers were anti-alpha trypsin (AAT),
neopterin, myeloperoxidase (MPO). The mRNA biomarkers were sucrase isomaltase (SI), caudal homeobox 1 (CDX1),
S100A8, and mucin (MUC12).
https://doi.org/10.1371/journal.pntd.0011112.g001
(see Eqs 1–4). Four score types were derived using different subsets of biomarkers: 1) a “Enter-
ocyte Integrity Score” consisting of SI, Cdx1 and mucin 12; 2) an “Acute Inflammation Score”
consisting of S100A8, MPO and AAT that all measured the neutrophil response; 3) a “Chronic
Inflammation Score” where neopterin is a proxy for the adaptive immune response; and 4) an
“Inflammation Score” consisting of S100A8, MPO, AAT and neopterin. Given the highly con-
taminated environments of these infants, the “Chronic Inflammation Score” can be treated as
a measure of frequently encountered pathogens, which often trigger an immune response. We
therefore use neopterin as a proxy for the adaptive immune response. In the compound scores
(Enterocyte Integrity, Acute Inflammation, and Inflammation), each of the biomarkers con-
tributed equally to the scores.
Health Score ¼ SI Quintile þ CDX1 Quintile þ Mucin12 Quintile

½0 4� ½0 4� ½0 4� ð1Þ
Maximum Score ¼ 12

Acute Inflammation Score ¼ S100A8 Quintile þ MPO Quintile þ AAT Quintile

½0 4� ½0 4� ½0 4� ð2Þ
Maximum Score ¼ 12
Chronic Inflammation Score ¼ Neopterin Quintile

½0 4� ð3Þ
Maximum Score ¼ 4
Inflammation Score ¼ S100A8 Quintile þ MPO Quintile þ AAT Quintile þ Neopterin Quintile
½0 4� ½0 4� ½0 4� ½0 4� ð4Þ
Maximum Score ¼ 16
For the creation of the “data derived” score, we first removed the outliers using the inter-
quartile range method (IQR) method where any observations that were more than 1.5 IQR
below Q1 or more than 1.5 IQR above Q3 were considered outliers and removed [34]. In total
13 samples were removed from the data set. The remaining values were standardized around
the mean. The PCA, was run using factor loading values of > 0.4 or < -0.4. Components were
selected that explained at least 80% of the cumulative proportion of the variance based on the
Kaiser-Guttman rule and the Scree test [35].
An infant’s score was arrived at by summing the product of the measured biomarker levels
and their respective factor loading values. Scores were then standardized around their means.
The score consisted of three components: an ‘Enterocyte Integrity’ score that measured the
physiological state of gut enterocytes and two inflammation scores that were classed as either,
‘Acute’, a measure of the innate neutrophil response or ‘Chronic’, a measure of the adaptive
immune response.
Fisher’s Exact tests were used to test for significant differences in the proportion of positive
samples (samples that tested positive for a pathogen) by age category and Wilcoxon Rank Sum
tests were used to test for significant differences in pathogen gene counts between age catego-
ries. Correlation among biomarkers was assessed using Spearman correlation coefficients.
Log-binomial models were used to determine the association between pathogen presence and
2-week diarrheal disease prevalence. The association between scores and stool pathogen gene
counts was assessed using linear models. Models were adjusted for the infant’s sex, age, and
the consistency of the stool sample. All analysis was done using R version 4.1.0. PCA’s were
performed using the FactoMineR package.
Results
Descriptive Statistics
Our sample of 136 were from infants between 6 months to 2 years (mean = 14.35 months,
SD = 4.80 months). Most were male (59%) and most were partially breastfed (77%). Only 7%
of the infants were exclusively breastfed at the time of sampling. Most stool samples provided
were formed (68%), with only 14% were classified as liquid.
The mean LAZ, WAZ, and WLZ of the infants in our sample were -0.89 (SD = 1.39), -0.23
(SD = 1.12), and 0.30 (SD = 1.20) respectively. Only 19, 6, and 2 infants had LAZ, WAZ, and
WLZ scores � -2 respectively (Table 1). A comparison of our biomarker values (Table 1) with
previously reported studies is provided in S3–S10 Tables.

Table 1. Demographic characteristics and descriptive statistics of infants and biomarkers.

N Mean SD Minimum Maximum Median (25th, 75th percentiles)
LAZ 136 -0.89 1.39 -4.75 1.98 -0.89 (-1.90, 0.12)
WAZ 136 -0.23 1.12 -2.56 3.03 -0.38,(-1.03, 0.43)
WLZ 136 0.30 1.20 -2.44 3.95 0.14 (-0.62, 1.22)
Age, months 136 14.35 4.80 6.00 24.00 14.00 (11.00, 18.00)
MPO, ng/mL 134 7080.21 12130.41 137.40 102498.70 2970 (1382.9, 8563.4)
Neopterin, nmol/L 93 1509.34 1183.41 4.40 4635.60 1166.3 (576.1, 2176.4)
AAT, ng/mL 119 596.42 617.67 14.58 3915.67 411.64 (205.38, 753.33)
SI 136 2.62 24.94 0.00 288.18 0.027 (0.00, 0.087)
CDX1 136 0.10 0.12 0.00 0.69 0.070 (0.027, 0.13)
MUC12 136 4.71 6.29 0.00 41.15 2.34 (1.15, 5.52)
S100A8 136 10.68 20.55 0.04 183. 17 4.48 (2.23, 13.45)
https://doi.org/10.1371/journal.pntd.0011112.t001
Stool pathogen frequency and gene counts in stool samples

Only the proportion of infants testing positive for Giardia was significantly different between
the two age categories, with 5% of infants 6 to 11 months old testing positive compared to 35%
of infants 12 months and older (p-value <0.001) (Fig 2 and S14 Table). Bacterial pathogens
were the most frequently detected pathogen type in both age groups. For example, 73% of
stool samples from infants 6 to 11 months old tested positive for the EPEC gene eae, as did
67% of stool samples from infants 12 months and older. However, there were no statistically
significant differences in the proportion of infants testing positive for a bacterial pathogen
between the two age categories. Norovirus GII was more commonly detected than norovirus
GI, detected in 45% of infants aged 6 to 11 months old and in 29% of infants 12 months and
older. As with bacterial pathogens, there were no statistically significant differences in the pro-
portion of infants testing positive for either norovirus GI or GII between the two age
categories.
Bacterial pathogens (specifically EAEC and EPEC) had the highest stool gene counts
(Table 2). In general, EAEC and EPEC gene counts were highest in infants aged 6 to 11 months
with marked decreases with age (p-value = 0.048 for EAEC). Norovirus GII stool gene counts
were higher than those for Norovirus GI and were lower in infants aged 12 months and older.
Among protozoa, Giardia stool gene counts were significantly higher in infants aged 12
months and older (p-value <0.001).
Spearman correlation coefficients between biomarkers

The strongest significant correlation between biomarkers was between MPO and AAT (Spear-
man Coefficient = 0.45) (Table 3). MPO also had weak significant correlations with S100A8
(Spearman Coefficient = 0.23) and mucin 12 (Spearman Coefficient = -0.20). The transcription
factor, Cdx1, was also weakly correlated with SI (Spearman Coefficient = 0.30) and S100A8
(Spearman Coefficient = -0.17).
Principal Component Analysis (PCA)

The PCA analysis indicated that 5 components were required to explain the variability in the
data (Table 4). Based on the biomarkers selected, each component represented a different
physiological score. Two components represented chronic inflammation: Chronic Inflamma-
tion A consisting of SI (0.44), AAT (0.49), and neopterin (0.50); and Chronic Inflammation
B consisting of Cdx1(-0.41), mucin 12 (-0.63), and neopterin (0.62). Two components

Fig 2. The number of infant stool samples testing positive for pathogenic gene markers in; (a) infants aged 6 to 11
months old (b), infants aged 12 months and older from informal settlements in Addis Ababa, Ethiopia.
Table 2. Geometric means of pathogen stool gene counts by age category. Statistically significant differences in pathogen loads by age category are bolded.
6 to 11 months (N = 40) 12 months and older (N = 96)
Pathogen Mean (95% CI) Mean (95% CI) p-value
EAEC_aaiC (gene copies/g) 4.75E+03 (3.54E+02,6.36E+04) 2.55E+02 (4.99E+01, 1.30E+03) <0.05
EPEC_eae (gene copies/g) 1.15E+04(8.55E+02, 1.55E+05) 1.60E+03 (3.54+02, 7.28E+03) 0.12
EPEC_bfpA (gene copies/g) 7.20E+03 (5.09E+02, 1.02E+05) 1.37E+03 (2.97E+02, 6.31E+03) 0.17
ETEC_STh (gene copies/g) 6.03E+00 (1.39E+00, 2.62E+01) 9.57E+00 (3.64E+00, 2.52E+01) 0.46
STEC_SltII (gene copies/g) 3.67E+00 (1.48E+00, 9.53E+00) 2.77E+00 (1.75E+00, 4.40E+00) 0.59
Shigella (gene copies/g) 5.27E+00 (1.52E+00, 1.83E+01) 2.21E+01(7.48E+00, 6.53E+01) 0.10
Campylobacter (gene copies/g) 5.66E+00 (1.30E+00, 2.46E+01) 1.61E+01 (5.76E+00, 4.48E+01) 0.17
Giardia (gene copies/g) 1.89E+00 (6.50E-01, 5.48E+00) 7.26E+01 (2.05E+01, 2.57E+02) <0.05
Cryptosporidium (gene copies/g) 1.70E+00 (9.90E-01, 2.92E+00) 3.44E+00 (1.80E+00, 6.55E+00) 0.16
Noro_GI 5.50E-01 (1.40E-01, 2.16E+00) 7.50 E-01 (2.80E-01, 1.98E+00) 0.84
Noro_GII 2.99E+01 (3.78E+00, 2.37E+02) 4.37E+00 (1.42E+00, 1.35E+01) 0.09

Table 3. Spearman correlation coefficients between biomarkers. Coefficients significant at the 0.05 level are bolded.
SI S100A8 MUC12 CDX1 AAT MPO Neopterin
SI 1.00
S100A8 -0.02 1.00
MUC12 0.07 0.17 1.00
CDX1 0.30 -0.17 0.08 1.00
AAT -0.12 0.02 0.04 -0.06 1.00
MPO -0.15 0.23 -0.20 -0.07 0.45 1.00
Neopterin -0.01 -0.19 -0.20 0.04 0.07 0.02 1.00
represented acute inflammation: Acute Inflammation A consisting of Cdx1 (-0.40), S100A8

(0.46), and MPO (0.59); and Acute Inflammation B consisting of Cdx1 (0.68) and S100A8
(0.49). The fifth component represented Enterocyte Integrity consisting of SI (-0.59), mucin
12 (-0.47), and S100A8 (-0.49).
Associations between stool pathogen gene counts and the theory driven score
Multivariable linear models were used to examine association between stool pathogen gene
counts and the three ‘theory driven’ scores (Fig 3). Noro GI was significantly associated with
both the Enterocyte Integrity Score and the Chronic Inflammation Score, after adjusting for all
stool pathogens, infant age, breast feeding status and stool consistency. Shigella was signifi-
cantly associated with Acute Inflammation Score. Score summary statistics and age group
stratified analysis are provided in S11 Table and S2 Fig respectively.
Associations between stool pathogen gene counts and the data derived PCA
score
Shigella stool gene counts were significantly associated with the Enterocyte Integrity Score as
well as the Acute Inflammation Score B. ETEC_STh was significantly associated with the
Acute Inflammation A score. Chronic Inflammation A and B scores were not significantly
associated with any pathogen stool gene counts. Score summary statistics and age stratified
analysis are provided in S12 Table and S3 Fig respectively.
Table 4. Factor loading values and the derivation of PCA score models. Factor loading values of > 0.4 or < -0.4 are bolded.
Full Panel PCA
Biomarker PC1: Chronic Inflammation PC2: Acute Inflammation PC3: Chronic Inflammation PC4: Enterocyte PC5: Acute Inflammation
A A B Integrity B
MPO 0.32 0.59 -0.09 0.14 0.36
AAT 0.49 0.33 -0.09 0.28 -0.27
Neopterin 0.50 0.00 0.62 0.01 0.07
SI 0.44 -0.39 0.15 -0.59 0.12
CDX1 0.22 -0.40 -0.41 0.31 0.68
MUC12 0.29 0.11 -0.63 -0.47 -0.28
S100A8 -0.28 0.46 0.10 -0.49 0.49
Standard deviation 1.22 1.16 1.08 0.99 0.92
Proportion of 0.21 0.19 0.17 0.14 0.12
Variance
Cumulative 0.21 0.40 0.57 0.71 0.83
Proportion

Fig 3. Associations between the theory driven score and stool pathogen gene counts; (a) associations between stool
pathogen gene counts and the Enterocyte Integrity Score, (b) associations between the overall Inflammation Score and
stool pathogen gene counts, (c) the associations between stool pathogen gene counts and Acute Inflammation, and (d)
associations between the Chronic Inflammation Score and stool pathogen counts.
Discussion
Our findings add to the growing body of evidence that enteric pathogen infections and their
hypothesized sequalae such as EED cannot be captured by a single catch-all definition but
rather result in multiple overlapping responses to pathogen exposure, differing in time and,
potentially, in physiological space [36]. Our results also confirm the findings of Manary and
colleagues in demonstrating that fecal mRNA transcripts hold promise for the measurement
of EED [20–23]. The utility of mRNA transcripts lies in their ability to measure specific cellular
processes, providing a more complete picture of the overall gut state. Given the time scales that
mRNA transcripts measure, they provide a more immediate snapshot of physiological and
immune responses. This may be especially valuable in understanding dynamic processes such
as pathogen infection and colonization. Given recent efforts to develop ‘panels’ of EED bio-
markers [37,38], coupling panels with metabolic and transcriptomic approaches will be espe-
cially valuable.
The use of two different scoring systems demonstrates that there are multiple ways to cap-
ture underlying pathogenic processes and scoring systems can be adapted to best suit the goals
of a study. Our ‘theory driven’ and ‘data derived’ (including a data derived score using only
MPO, AAT and, neopterin (See S13 Table and S4 Fig)) scores largely agree especially regarding
how they capture the associations between stool pathogen gene counts and acute and chronic
inflammation. Notably, both approaches demonstrate how Shigella infection not only elicits a
strong neutrophil response (Figs 3, 4, and S4), but also profoundly affects enterocyte integrity
as shown by the negative association between Shigella gene stool gene counts and the Entero-
cyte Integrity Score in the “data derived” score (Fig 4). We believe that the biomarkers detect

Fig 4. Associations between the data derived score and stool pathogen gene counts; (a) associations between Chronic
Inflammation Score A (SI, AAT, and neopterin) and stool pathogen gene counts, (b) associations between Chronic
Inflammation Score B (Cdx1, mucin12, and neopterin) and stool pathogen gene counts (c) associations between
Enterocyte Integrity (SI, mucin12, and S100A8) and stool pathogen gene counts, (d) associations between stool
pathogen gene counts and the Acute Inflammation Score A (Cdx1, S100A8, and MPO), and (e) associations between
stool pathogen gene counts and Acute Inflammation Score B (Cdx1 and S100A8).
the massive inflammatory response associated with apoptotic macrophages, infiltration of

polymorphonuclear leukocytes and epithelial destruction that are characteristic of shigellosis
[13]. Our ability to capture the impact of Shigella infection on enterocyte integrity is due to the
inclusion of the mRNA transcript biomarkers which provide cell specific readouts, which in
the case of shigellosis is the destruction of gut enterocytes. Strikingly, however, the enterocyte
integrity scores did not agree between the two scoring systems. Given the complexity of EED
as well as the poorly understood and complex relationships between the different biomarkers,
the “theory driven” score may have subsumed multiple etiologic processes into a single bio-
marker, especially when using a small biomarker panel and sample.
Our work also shows that pathogenic E. coli carriage may be the norm in highly contami-
nated settings. This is similar to other studies that have reported that a significant proportion
of children continue to have sub-clinical pathogen carriage [39–42]. EPEC, the most detected
pathogen, was associated with both acute and chronic inflammation processes. EPEC infection
results in localized destruction of the intestinal brush border and distortion of the apical enter-
ocyte membrane [43]. EPEC infection has been proposed to occur in four stages: 1) EPEC cells
express 3 proteins (bundle-forming pili (Bfp), intimate adhesin intimin, and short surface-
associated filaments (EspA filaments) [43]; 2) EPEC cells adhere to epithelial cells via Bfp and
EspA filaments and cause depolymerization of actin and the loss of microvilli [43]; 3) EspA fil-
aments are lost from the bacterial cell surface and adhesin binding results in the accumulation
of actin and other cytoskeletal elements beneath the site of bacterial adherence [43]; 4) bacte-
rial attachment results in massive accumulation of cytoskeletal elements resulting in the loss of
tight-junction integrity and mitochondrial function that leads to both electrolyte loss and

eventual cell death [43]. Future research could focus on whether biomarker levels are associ-
ated with different stages of EPEC infection.
The prolonged and asymptomatic carriage of pathogens such as EPEC, necessitates the
need for diagnostic techniques that can distinguish between symptomatic and sub-clinical
infections. Though we did not see any significant differences in scores between infants who
had diarrhea in the past 2-weeks and those who had not (S15 Table), our work shows that
there are measurable physiological processes associated with infection, regardless of symptom
presentation. For example, though the detection of Shigella was not associated with 2-week
diarrheal disease prevalence, we were able to measure physiological impacts of Shigella infec-
tion, including enterocyte damage and acute inflammation. We hope that future work can fur-
ther explore how curated biomarker panels can be used to distinguish between symptomatic
and sub-clinical infections, which the small sample size and cross-sectional nature of our study
precluded us from doing.
To our knowledge, no other studies have characterized mRNA transcript-based biomarkers
in an urban population of infants. Compared to previously reported scores from rural popula-
tions of infants in Malawi and Sierra Leone, our mRNA transcript expression levels would sug-
gest better gut health in our infants (S6–S10 Tables). Fecal protein biomarker measurements
on the hand were in broad agreement with what has previously been reported (S3–S5 Tables).
Mean biomarker levels for MPO and neopterin, are marginally lower compared to other stud-
ies, while AAT levels were similar (7080.21ng/mL, 1509.34nmol/L, and 596.42 ng/mL respec-
tively). AAT is a modulator of the neutrophil response and inhibits MPO, and the higher ratio
of AAT to MPO in our sample compared to that reported in other studies, may be indicative
of a more controlled immune response in our samples [44]. The differences in agreement
between the mRNA transcript and stool protein markers suggests that mRNA transcripts and
protein biomarkers may measure responses that differ physiologically and temporally. Though
at the protein level infants appeared broadly similar to populations reported elsewhere
[9,15,45–47], the differences between our study and previously reported studies at the transcript
level may indicate that responses to stressors vary by geography and population even though
the end result is broad systemic inflammation. Specific biomarker panels to measure both spe-
cific cellular and physiologic processes and relevant EED endpoints will be especially valuable in
the establishing causality of the inflammatory end-states. Recent findings, such the establish-
ment of a causal relationship between the duodenal microbiota and growth faltering when com-
bined with metabolomics should aid biomarker selection, both at the mRNA transcript and
protein levels [6].
Overall, our findings add to a growing body of literature showing that the evaluation of
physiological processes resulting in systemic inflammatory endpoints will require more
nuanced approaches and that a standard set of diagnostic criteria may be hard to define.
Though the selected biomarkers could broadly measure physiological and immunological pro-
cesses, a more detailed understanding of the continuum of physiological responses ranging
from transient immune responses to chronic systemic dysregulation is necessary. In addition,
given that a goal of our study was to evaluate whether biomarker panels can measure gut spe-
cific processes, we did not specifically evaluate inter-biomarker associations beyond correla-
tions. Future work that addresses some of our limitations such as small sample size and lack of
temporal follow up, will be needed to better evaluate any relationships between biomarkers.
The development of biomarker panels can provide valid measures of overall gut health, but
also to provide cell-specific measures that inform our understanding of the physiological
responses to transient insults, long-term asymptomatic pathogen carriage, and chronic end
states such as EED. Such panels are valuable tools that complement existing direct measures of

EED such as the L:M test in understanding the impact of pathogenic stressors on long-term
outcomes in infants in low resource settings.
Supporting information
S1 Text. Droplet Digital PCR Detection of mRNA Transcripts. Protocol for the detection of
mRNA transcripts using ddPCR.
(DOCX)
S2 Text. Droplet Digital PCR Quantification of Stool Pathogen Loads. Protocol for the
quantification of stool pathogen gene loads using ddPCR.
(DOCX)
S1 Table. Pathogen specific gene targets used for the quantification of stool pathogen
loads. Gene targets and ddPCR Supermix used for the quantification of stool pathogen gene loads.
(DOCX)
S2 Table. Derivation of the theory driven histological score based on indicators laid out in
Liu et.al (2020). Explanation of the derivation of the theory driven score.
(DOCX)
S3 Table. Comparison of study myeloperoxidase levels with previously reported levels.
Table comparing study measured myeloperoxidase levels with levels reported in a subset of
previous studies.
(DOCX)
S4 Table. Comparison of study AAT levels with previously reported levels.
Table comparing study measured AAT levels with levels reported in a subset of previous stud-
ies.
(DOCX)
S5 Table. Comparison of study neopterin levels with previously reported levels.
Table comparing study measured neopterin levels with levels reported in a subset of previous
studies.
(DOCX)
S6 Table. Comparison of study transcript expression levels and expression levels previ-
ously reported in Malawian infants with varying L:M ratios. Table comparing study tran-
script levels with transcript levels from Malawian infants with different L:M ratios.
(DOCX)
S7 Table. Comparison of study transcript expression levels previously reported expression
levels in Malawian infants. Table comparing study transcript levels with transcript levels in
two previous studies in Malawian infants.
(DOCX)
S8 Table. Comparison of study transcript expression levels with expression levels in Mala-
wian infants aged less than 12 months and 12–61 months. Comparison of study transcript
levels with age stratified transcript levels from Malawian infants and young children.
(DOCX)
S9 Table. Comparison of study transcript expression levels with those of Malawian infants
with severe EED and no to moderate EED. Comparison of study transcript levels with levels
in Malawian infants with and without EED.
(DOCX)

S10 Table. Comparison of study transcript expression levels with those of Sierre Leonean
Infants. Comparison of study transcript levels with transcript levels in Sierra Leonean infants.
(DOCX)
S11 Table. Summary statistics of the theory driven scores.
(DOCX)
S12 Table. Summary statistics of the data derived scores.
(DOCX)
S13 Table. Factor loading values and score types for PCA derived using only myeloperoxi-
dase, AAT and neopterin.
(DOCX)
S14 Table. Number of samples testing positive for each pathogen.
(DOCX)
S15 Table. Comparison of Scores by 2-week diarrheal disease prevalence.
(DOCX)
S16 Table. Unadjusted associations between pathogen prevalence and 2-week diarrheal
disease prevalence.
(DOCX)
S1 Fig. Correlation between age and biomarker levels in the full sample.
(PDF)
S2 Fig. Associations between the theory driven score and age stratified stool pathogen gene
counts.
(PDF)
S3 Fig. Associations between the data derived score and age stratified stool pathogen gene
counts.
(PDF)
S4 Fig. Associations between the protein biomarker-based data derived score and stool
pathogen gene counts.
(PDF)
S5 Fig. Associations between the protein biomarker-based data derived score and stool
pathogen gene counts in a) infants aged 6–11 months and b) in infants aged 12 months
and older.
(PDF)
Acknowledgments
We would like to thank the people and communities of Addis Ababa for welcoming us into
their homes and making this study possible. We are also grateful to the Ethiopian Institute for
Public Health for providing us with lab space and equipment for some of the assays.
Author Contributions
Conceptualization: Leon M. Espira, Gwenyth O. Lee, Kaleab Baye, Joseph N. S. Eisenberg.
Data curation: Leon M. Espira.

Formal analysis: Leon M. Espira.

Funding acquisition: Gwenyth O. Lee, Andrew D. Jones, Nancy G. Love, Joseph N. S.
Eisenberg.
Investigation: Leon M. Espira.
Methodology: Leon M. Espira, Gwenyth O. Lee.
Project administration: Leon M. Espira.
Supervision: Andrew D. Jones, Nancy G. Love, Joseph N. S. Eisenberg.
Writing – original draft: Leon M. Espira, Gwenyth O. Lee, Kaleab Baye, Andrew D. Jones,
Nancy G. Love, Joseph N. S. Eisenberg.
Writing – review & editing: Leon M. Espira, Gwenyth O. Lee, Kaleab Baye, Andrew D. Jones,
Nancy G. Love, Joseph N. S. Eisenberg.
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PLOS NEGLECTED TROPICAL DISEASES

Stool biomarkers as measures of enteric

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 1 / 18

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 2 / 18

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 3 / 18

Materials and methods

Stool sample processing

ELISA protein biomarker measurements

Nucleic acid extraction

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 4 / 18

Droplet digital PCR detection of mRNA transcripts

Droplet digital PCR quantification of stool pathogen gene counts

Droplet digital PCR data processing

Development of Physiological Scores and Statistical Analysis

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 5 / 18

Health Score ¼ SI Quintile þ CDX1 Quintile þ Mucin12 Quintile

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 6 / 18

Acute Inflammation Score ¼ S100A8 Quintile þ MPO Quintile þ AAT Quintile

Chronic Inflammation Score ¼ Neopterin Quintile

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 7 / 18

Table 1. Demographic characteristics and descriptive statistics of infants and biomarkers.

Stool pathogen frequency and gene counts in stool samples

Spearman correlation coefficients between biomarkers

Principal Component Analysis (PCA)

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 8 / 18

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 9 / 18

represented acute inflammation: Acute Inflammation A consisting of Cdx1 (-0.40), S100A8

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 10 / 18

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 11 / 18

the massive inflammatory response associated with apoptotic macrophages, infiltration of

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PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011112 February 21, 2023 15 / 18

Formal analysis: Leon M. Espira.

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