What Is Known
- In pediatric cohort studies, enteroaggregative Escherichia coli was frequently detected in developing countries;
- Enteroaggregative E coli was associated with growth deficits in these children;
- The potential impact of enteroaggregative E coli subclinical infections and the presence of co-infections on child growth remain unclear.
What Is New
- Isolated subclinical enteroaggregative E coli infection does not influence child growth in the first six months of life;
- Increased pathogen enteroaggregative E coli coinfection is negatively associated with delta weight-for-length and weight-for-age z scores, which are partially dependent on the presence of enteroaggregative E coli.
- These data emphasize the importance of subclinical enteroaggregative E coli coinfections in early childhood.
Escherichia coli are an important cause of enteric infections, and the following 5 enterovirulent types have been identified: enterotoxigenic E coli, Shiga toxin-producing E coli, enteroinvasive E coli, enteropathogenic E coli, and enteroaggregative E coli (EAEC) (1–3). The multisite birth cohort study, Malnutrition and Enteric Diseases (MAL-ED), involved intensive community surveillance for diarrhea and nondiarrheal stools over the first 2 years of life from 8 sites in South America, Africa, and Asia, showed that EAEC were frequently detected in children with and without diarrhea (4). These results were consistent with early data reported in a small cohort study in Fortaleza CE, Brazil (5). The EAEC pathogen burden in the MAL-ED birth cohort study was associated with growth deficits in these children (The MAL-ED contributors. Factors affecting growth velocity and risk factors for stunting in the first 24 months of life: results from the MAL-ED study. MAL-ED major paper in preparation to submit soon). However, the potential impact of EAEC associated with coinfections in early infancy on child growth remains unclear. The MAL-ED birth cohort study also reported that both the incidence of diarrhea and the number of pathogens detected per stool increased markedly during the first year of life (4). Two or more pathogens were identified in 41% (2999) of diarrhea samples and 29% (7046) of nondiarrheal samples, suggesting that coinfections are common in these children in their first year of life. Therefore, we examined subclinical or “silent” EAEC infections alone and in combination with other pathogens and their associations with child growth in the first 6 months of life.
Early studies have shown that EAEC is an inflammatory pathogen and that growth deficits occur in children when the bacterium is acquired, with or without diarrheal symptoms (6,7). The potential impact of subclinical EAEC infections and the presence of coinfections on the pathobiology of EAEC infections and effects on child growth remain unclear.
We hypothesized that maternal education, birth weight, breast-feeding, and socioeconomic status (SES) will increase the risk of acquiring asymptomatic EAEC alone or with other enteric pathogens in early infancy, leading to gut inflammation and impaired growth. The aim of the present study was thus to understand the risk factors, gut integrity, inflammation, and innate immune responses associated with EAEC or in combination with other enteric pathogens and their impact on growth in the first 6 months of life across all 8 sites in the MAL-ED multisite birth cohort study.
This study was conducted across 8 locations: Dhaka, Bangladesh (BGD); Fortaleza, Brazil (BRF); Vellore, India (INV); Bhaktapur, Nepal (NEB); Loreto, Peru (PEL); Naushero Feroze, Pakistan (PKN); Venda, South Africa (SAV); and Haydom, Tanzania (TZH). A detailed description of the MAL-ED study location, demography, and SES has been reported elsewhere (8–15).
Study Design, Population, and Ethical Approval
In this longitudinal birth cohort study, infants up to 2 years of age were followed-up in each of the 8 study sites; this report included data collected during the first 6 months of life. The overall design of the project has been described in detail elsewhere (16–23). The study and consent protocols were approved by the local institutional review board (IRB) at all sites and the collaborating institution IRBs. Written informed consent was obtained from the parent or guardian of every child. We enrolled infants within 17 days of birth between November 2009 and February 2012.
Surveillance and Stool Collection
Surveillance was performed during twice-weekly household visits. Caregivers responded to a standardized questionnaire designed to collect data regarding daily symptoms of cough, fever, vomiting, diarrhea, and medication use. We investigated nondiarrheal specimens that were collected during the surveillance between 1 and 6 months of age. The overall surveillance methods used in the MAL-ED cohorts have been described in detail elsewhere (16).
Maternal Education, Birth Weight, and Breast-feeding Variables, and Socioeconomic Status
Questionnaires were developed to collect information about child anthropometrics, child care, characteristics of the mother or caregiver, household, people usually sleeping in the house, sources of water, toilet facilities, average monthly income, and other related parameters. For SES determination, we used a standardized SES questionnaire applicable to the MAL-ED cohorts (24). The defined variables and their parameterizations are shown in Supplementary Table 1 (Supplemental Digital Content 1, https://links.lww.com/MPG/B92).
The study protocol used a standard recumbent length measuring board (Schorr Productions, Olney, MD) to measure the monthly length of all enrolled children to the nearest 0.1 cm. Digital scales were also used monthly to measure weight to the nearest 100 g. The weight-for-age (WAZ), length-for-age (LAZ), and weight-for-length (WLZ) z scores were calculated using the World Health Organization Multi-Country Growth Reference Study (25).
Microbiology and Stool Testing
The nondiarrheal specimens were analyzed in accordance with a standardized microbiology protocol, which was implemented at all study sites. The protocol has previously been described in detail (20). Briefly, we selected a pool of 5 lactose-fermenting colonies resembling E coli and characterized them for virulence genes using a multiplex polymerase chain reaction assay. Details of the virulence genes selected for the polymerase chain reaction probes are presented in the references of the article by Houpt et al (20).
Gut Function Integrity, Immune, and Inflammatory Biomarkers
The lactulose:mannitol test was used to evaluate intestinal permeability, malabsorption, and damage and was administered to children at 3 and 6 months. The average of these 2 measurements used for the analysis. Lactulose and mannitol were measured as previously described (26).
Three additional biomarkers were also measured monthly in nondiarrheal stools between 0 and 6 months of age and the average of these measurements used for this analysis. These included alpha-1-antitrypsin (A1AT), myeloperoxidase (MPO), and neopterin (NEO) (26).
The MAL-ED cohort study used a standardized protocol and data collection tools (27). On-site training, quality assurance, and quality control protocols enabled this study to maintain a harmonized, quality database for analysis. The data were entered using a double data entry system in Microsoft Access (Microsoft Corporation, Redmond, WA).
We evaluated cumulative EAEC infection alone and in combination with any other enteric pathogen in monthly nondiarrheal stools from asymptomatic children across 8 MAL-ED sites in Asia, Africa, and Latin America. Children were selected when they had ≥90% active surveillance (Surveillance Assessment Form) for 0 to 6 months, had collected a Follow-up Socioeconomic status form for 0 to 6 months, and had stool samples with a complete microbiology workup.
Pathogen Coinfections and Outcome Variables
To determine the impact of EAEC infection alone and in combination with any other enteric pathogen coinfection, we divided the cohort children into 7 groups based on the cumulative monthly stool detection of enteric pathogens as follows: children with no pathogen detection in every stool collected; children with EAEC in any stool collected; children with EAEC and 1 other pathogen; children with EAEC and 2 other pathogens; children with EAEC and 3 or more other pathogens; children with 1 or 2 pathogens other than EAEC; and children with 3 or more pathogens other than EAEC.
The major outcome variables were WAZ, length-for-age, and WLZ deltas z scores (0–6 months). The secondary outcome variables included the average 0 to 6 months’ gut function, inflammation, and innate immune response marker association in the study groups. Categorization of groups based on cumulative EAEC infection alone or coinfection with any other enteric pathogen enabled us to assess the impact of EAEC alone or in combination with other enteric pathogens on child growth.
Chi-square or Fisher exact tests were performed to compare categorical variables between the 7 groups defined above. Student t tests for normally distributed data and Kruskal-Wallis tests for non-normally distributed data were used to compare continuous variables between these groups.
We performed a mixed-effects linear regression analysis on child growth in the first 6 months of life. The differences in WAZ, WLZ, and LAZ z scores between enrollment and 6 months were regressed against EAEC exposure with and without other enteric pathogens. We included model covariates based on biologic plausibility that included child sex, weight, and/or length at enrollment, an indicator of household food insecurity, and proportion of days of breast-feeding, symptoms of acute lower respiratory infection, and antibiotic use. Definitions of the surveillance covariates are provided in Supplementary Table 1 (Supplemental Digital Content 1, https://links.lww.com/MPG/B92). The model included a random intercept on the study site to account for unexplained variation in the model due to site variability.
Statistical analysis was conducted using IBM SPSS Statistics for Windows, version 20.0 (IBM Corp, Armonk, NY). Regression and plots were performed in R v.3.2.2 using the “lme4” package. P values <0.05 were considered statistically significant.
Participant Enrollment and Selection
Across 8 study sites, 2145 children were enrolled on a rolling basis between November 2009 and February 2012 and longitudinally followed. Of these, stool samples with a complete microbiology workup were available for 1695 children. Finally, 1684 children who underwent a minimum of 90% of the active surveillance assessments between 0 and 6 months and whose parents or legal guardians had answered an SES questionnaire during this time were included in the final analysis. The 1684 children provided 8216 surveillance stools up to 6 months of age. The groups of children and details of the stools provided for analysis are shown in Table 1.
Association of Potential Risk Determinants With Subclinical Enteroaggregative Escherichia coli Infection Alone or in Combination With Other Pathogens
Table 1 summarizes the categorical risk variables associated with subclinical EAEC infections through the first 6 months of age. There were no differences in sex or age between the groups tested. The proportions of children with birth weights <2500 g were also similar among all groups. Mothers with <6 years of schooling and no suitable drinking water sources were significantly more common for children with subclinical EAEC infections with ≥3 other pathogens compared to all other groups of children. Both subclinical infections with EAEC plus ≥3 other pathogens and ≥3 pathogens without EAEC were significantly more common in children with inadequate sanitation compared to the other pathogen groups. Food insecurity was present in a smaller proportion in the group of children with subclinical EAEC infection alone compared to no pathogens. Children with subclinical EAEC infection with only 1 other pathogen had less food insecurity compared to EAEC with 2 other pathogens. Table 2 shows the quantitative risk variables associated with subclinical EAEC infections through the first 6 months of age. Birth weights, mothers’ years of schooling, and monthly incomes were similar across all groups of children. The SES measured via the asset score was lower in the group with EAEC combined with ≥3 other pathogens compared to the scores for all other groups of children. The SES was also lower in the group with EAEC combined with 1 or 2 other pathogens compared to those in the following groups: no pathogen, EAEC alone, and 1 or 2 pathogens without EAEC. Finally, the SES was lower in the group with ≥3 pathogens without EAEC than in the nonpathogens group. A similar significant trend was also observed for SES measured via the water/sanitation, household assets, maternal education, and household income as shown in Table 2. The proportion of days of antibiotic use was significantly higher in the group of children with EAEC subclinical infection with ≥3 other pathogens than in all other groups. The proportion of breast-feeding days was significantly lower in the group with EAEC subclinical infections with ≥3 other pathogens compared to those in all other groups except for ≥3 pathogens without EAEC. The proportion of breast-feeding days was also lower in the group with EAEC combined with 2 other pathogens compared to that in the nonpathogen group.
Gut Function Barrier Integrity, and Immune and Inflammatory Biomarkers
Gut function as measured by the adjusted z scores of the percentages of lactulose and mannitol excreted in the urine and lactulose:mannitol ratios were similar across all groups of children (Table 3). Stool MPO concentrations were significantly higher in the subclinical EAEC infection with ≥3 other pathogens group than that in the nonpathogen group. MPO concentration was also higher in children with EAEC with 2 other pathogens than in the nonpathogen group. Lower concentrations of stool neopterin were found in the groups of children with subclinical EAEC infection with ≥3 other pathogens compared to all other groups of children, except ≥3 pathogens without EAEC; children with EAEC with 2 other pathogens (vs no pathogens and vs 1 or 2 pathogens without EAEC); children with ≥3 pathogens without EAEC (vs no pathogens and vs 1 or 2 pathogens without EAEC). Stool alpha-1-antitrypsin marker concentrations were comparable across all groups of children.
Effect of Subclinical Enteroaggregative Escherichia coli Infections Alone or Combined With Any Other Pathogen on Cumulative Child Growth
Cumulative growth was measured up to 6 months of age. Table 4 and Supplementary Figure 1 (Supplemental Digital Content 2, https://links.lww.com/MPG/B93) summarize the delta z scores WLZ, WAZ, and LAZ. Children with EAEC with ≥3 other pathogens showed an impaired delta WLZ z score compared to all other groups, except for EAEC with 2 other pathogens. The delta WAZ z score was also significantly lower in the EAEC with ≥3 other pathogens group compared to all other groups, except for EAEC with 2 other pathogens and nonpathogens (borderline; P = 0.056).
The regression results of the association between EAEC exposure, in the absence of other pathogens, with growth over the first 6 months of life were not statistically significant (Fig. 1). However, when EAEC was associated with coinfection with 1, 2, or 3 additional pathogens, decreased delta WLZ and WAZ were observed (Fig. 1). When EAEC was associated with coinfection with 3 or more additional pathogens, WLZ was −0.244 z scores lower than that in children with no pathogens present (P < 0.05, Fig. 1). This association was not maintained when 3 or more pathogens were present in the absence of EAEC (Fig. 1).
Prevalence of Subclinical Enteric Coinfections With Enteroaggregative Escherichia coli
Across the cohort's study sites, we observed that the groups of children with subclinical EAEC infection with 2 or ≥3 other pathogens were strongly associated with impaired physical growth, as measured by WLZ and WAZ delta z scores. Therefore, we explored the prevalence of subclinical enteric coinfections with EAEC in these 2 groups of children. Figure 2A shows that the highest prevalence of enteric coinfection involved Campylobacter spp and atypical enteropathogenic Escherichia coli in children with EAEC and 2 pathogens. Campylobacter spp, enterotoxigenic E coli thermo-labile toxin−producing, and Cryptosporidium spp were the most prevalent coinfection pathogens in children with EAEC with ≥3 other pathogens. Figure 2B shows only the enteric pathogens with >1% of the cumulative prevalence up to 6 months of age.
In nondiarrheal stool samples, subclinical EAEC infection alone was not significantly associated with child growth between enrollment and 6 months of age. However, increasing pathogen codetection with EAEC was negatively associated with decreased delta WAZ and WLZ from 0 to 6 months. Compared to children with no pathogens detected, the mean delta WLZ was approximately 0.25 lower in children with EAEC and 3 or more pathogens (P < 0.05) and the mean delta WAZ was approximately 0.16 lower in children with EAEC and 3 or more pathogens (P > 0.05). There was no clear trend in the correlation between EAEC exposure and LAZ with or without pathogen codetection. However, early cohort studies reported an association between EAEC subclinical infection and child growth impairment (The MAL-ED contributors. Factors affecting growth velocity and risk factors for stunting in the first 24 months of life: results from the MAL-ED study. MAL-ED major paper in preparation to submit soon). A previous analysis of EAEC in the MAL-ED study showed that consistent detection of EAEC across the first 2 years of life was associated with linear growth deficits at 2 years of age (28). This previous work, however, did not explore coinfection. In addition, increasing pathogen EAEC codetection was negatively associated with WLZ and WAZ in the present study. This effect was not observed in other pathogen coinfections in the absence of EAEC. These results suggest a pathobiological interaction between EAEC and other pathogens, resulting in growth impairment at a critical stage of early development. A recent study using microbiota from a Malawian birth cohort in an undernourished donor community administered to recipient gnotobiotic mice produced a growth deficit compared to that from a healthy donor community (29). These data suggest the hypothesis that gut microbiota immaturity together with enteric copathogens including EAEC may impact growth development or have other long-term consequences, a possibility that warrants further study.
Few studies have evaluated isolated EAEC subclinical infection with determinant variables; several have reported an association between inadequate or contaminated food and water and EAEC infection without examining pathogen codetection (30–32). Comparable associations between EAEC infection and poor hygiene and host immunosuppression have been reported (33). Therefore, we limited the focus of this work to the variables associated with EAEC coinfections in the current literature.
Consistent with recent publications from the MAL-ED birth cohort and BRF site case-control MAL-ED studies, EAEC alone and all the other groups of children presented with high urine LM-Z ratio and fecal MPO, A1AT, and NEO biomarker levels, suggesting environmental enteropathy disease (26,34). MPO concentration was significantly higher in children with EAEC and 3 or more pathogens and in children with EAEC with co-detection of 2 other pathogens compared to the concentrations in children in which no pathogens were detected. These results suggest an interaction effect of EAEC with 2 or more pathogens on gut inflammatory responses but not in the absence of EAEC with 2 or more pathogens. Studies have shown colonization of EAEC without overt symptoms of diarrhea; however, these studies did not evaluate pathogen codetection (6,7). Furthermore, the studies concluded that EAEC was an inflammatory pathogen and that infection resulted in growth deficit even without overt symptoms of diarrhea (6,7). The present report suggests for the first time that early childhood growth deficits are associated with EAEC and coinfections in an asymptomatic cohort of children. The pathobiology of subclinical EAEC infection with pathogen codetection appears to be more complex than previously thought; therefore, further studies are required to examine this in detail. NEO concentration was higher in the nonpathogen group compared to that in EAEC group with 2 or more other pathogens and in the group with 3 or more pathogen codetection without EAEC. These data suggest greater protection in the gut immune responses in the control group compared to the other groups of children.
This study has several limitations. First, despite monthly stool analysis over 6 months, subclinical pathogen codetection could have been missed between stool sample collections. Second, the study group definition and design analyses do not account for the duration of subclinical infections, even though we had a sense of repeated detection and quantitative specific pathogen detection. These are relevant parameters to consider for further studies on the pathobiology and impact of isolated subclinical EAEC and codetected pathogen infections on physical growth in children. The study also has several strengths. First, the MAL-ED multicenter study collects comprehensive information on determinant variables such as maternal and child care characteristics, sanitation, hygiene, and SES, which enable assessment of the association or influence of the determinant variables on child nutritional status after 6 months of follow-up. Second, the study also collects information on important biomarkers, allowing for examination of key potential pathobiological association with the subclinical isolated EAEC and coinfections. Third, the MAL-ED study allowed co-infection analysis to examine the potential interaction associations of isolated EAEC and pathogen coinfection with child growth.
In conclusion, silent (ie, acutely asymptomatic) enteric coinfections with EAEC and other pathogens in the first 6 months of life were associated with significant growth deficits (decreasing delta WLZ z score). Further study of the potential associations and mechanisms of infection with EAEC alone and coinfection with other potential pathogens is warranted, and potential strategies to prevent growth deficit or other long-term consequences are required.
The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project (MAL-ED) is carried out as a collaborative project supported by the Bill & Melinda Gates Foundation, the Foundation for the NIH, and the National Institutes of Health/Fogarty International Center. The authors thank the staff and participants of the MAL-ED Network for their important contributions.
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