Gwenyth Lee

Fecal markers of intestinal inflammation and permeability associated with the subsequent acquisition of linear growth deficits in infants.

Enteric infections are associated with linear growth failure in children. To quantify the association between intestinal inflammation and linear growth failure three commercially available enzyme-linked immunosorbent assays (neopterin [NEO], alpha-anti-trypsin [AAT], and myeloperoxidase [MPO]) were performed in a structured sampling of asymptomatic stool from children under longitudinal surveillance for diarrheal illness in eight countries. Samples from 537 children contributed 1,169 AAT, 916 MPO, and 954 NEO test results that were significantly associated with linear growth. When combined to form a disease activity score, children with the highest score grew 1.08 cm less than children with the lowest score over the 6-month period following the tests after controlling for the incidence of diarrheal disease. This set of affordable non-invasive tests delineates those at risk of linear growth failure and may be used for the improved assessments of interventions to optimize growth during a critical period of early childhood.

Assessment of Environmental Enteropathy in the MAL-ED Cohort Study: Theoretical and Analytic Framework

Individuals in the developing world live in conditions of intense exposure to enteric pathogens due to suboptimal water and sanitation. These environmental conditions lead to alterations in intestinal structure, function, and local and systemic immune activation that are collectively referred to as environmental enteropathy (EE). This condition, although poorly defined, is likely to be exacerbated by undernutrition as well as being responsible for permanent growth deficits acquired in early childhood, vaccine failure, and loss of human potential. This article addresses the underlying theoretical and analytical frameworks informing the methodology proposed by the Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study to define and quantify the burden of disease caused by EE within a multisite cohort. Additionally, we will discuss efforts to improve, standardize, and harmonize laboratory practices within the MAL-ED Network. These efforts will address current limitations in the understanding of EE and its burden on children in the developing world.

Geophagy is associated with environmental enteropathy and stunting in children in rural Bangladesh.

There is a growing body of literature indicating an association between stunting and environmental enteropathy (EE), a disorder thought to be caused by repeated exposures to enteric pathogens. To investigate the relationship between exposure to enteric pathogens through geophagy, consumption of soil, EE, and stunting, we conducted a prospective cohort study of 216 children under 5 years of age in rural Bangladesh. Geophagy was assessed at baseline using 5 hour structured observation and caregiver reports. Stool was analyzed for fecal markers of intestinal inflammation: alpha-1-antitrypsin, myeloperoxidase, neopterin (all three combined to form an EE disease activity score), and calprotectin. Eighteen percent of children had observed geophagy events by structured observation and 28% had caregiver reported events in the past week. Nearly all households had Escherichia coli (97%) in soil, and 14% had diarrheagenic E. coli. Children with caregiver-reported geophagy had significantly higher EE scores (0.72 point difference, 95% confidence interval [CI]: 0.01, 1.42) and calprotectin concentrations (237.38 μg/g, 95% CI: 12.77, 462.00). Furthermore, at the 9-month follow-up the odds of being stunted (height-for-age z-score < -2) was double for children with caregiver-reported geophagy (odds ratio [OR]: 2.27, 95% CI: 1.14, 4.51). These findings suggest that geophagy in young children may be an important unrecognized risk factor for EE and stunting.

Symptomatic and asymptomatic Campylobacter infections associated with reduced growth in Peruvian children.

Background Although diarrheal illnesses are recognized as both a cause and effect of undernutrition, evidence for the effect of specific enteropathogens on early childhood growth remains limited. We estimated the effects of undernutrition as a risk factor for campylobacteriosis, as well as associations between symptomatic and asymptomatic Campylobacter infections and growth. Methodology/Principal Findings Using data from a prospective cohort of 442 children aged 0–72 months, the effect of nutritional status on the incidence of Campylobacter infection was estimated using uni- and multivariate Poisson models. Multivariate regression models were developed to evaluate the effect of Campylobacter infection on weight gain and linear growth. Overall, 8.3% of diarrheal episodes were associated with Campylobacter (crude incidence rate = 0.37 episodes/year) and 4.9% of quarterly asymptomatic samples were Campylobacter positive. In univariate models, the incidence of Campylobacter infection was marginally higher in stunted than non-stunted children (IRR 1.270, 95% CI (0.960, 1.681)(p = 0.095). When recent diarrheal burdens were included in the analysis, there was no difference in risk between stunted and unstunted children. Asymptomatic and symptomatic Campylobacter infections were associated with reduced weight gain over a three-month period (65.5 g (95% CI: −128.0, −3.0)(p = 0.040) and 43.9 g (95% CI:−87.6, −1.0)(p = 0.049) less weight gain, respectively). Symptomatic Campylobacter infections were only marginally associated with reduced linear growth over a nine month period (−0.059 cm per episode, 95% CI: −0.118, 0.001)(p = 0.054), however relatively severe episodes were associated with reduced linear growth (−0.169 cm/episode, 95% CI −0.310, −0.028)(p = 0.019). Conclusions/Significance Our findings suggest that Campylobacter is not as benign as commonly assumed, and that there is evidence to support expanding the indications for antibiotic therapy in campylobacteriosis in children.

Detection of Campylobacter in Stool and Determination of Significance by Culture, Enzyme Immunoassay, and PCR in Developing Countries

ABSTRACT Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level.

Comparative effects of vivax malaria, fever and diarrhoea on child growth.

BACKGROUND The adverse impact of Plasmodium vivax on child health beyond acute febrile illness is poorly studied. The effect of vivax malaria on child growth was evaluated and compared with diarrhoeal disease and non-specific fever. METHODS Using data from a 43-month longitudinal cohort of children 0-72 months of age (n = 442) in the Peruvian Amazon, ponderal and linear growth velocities over 2-, 4- and 6-month periods were examined using longitudinal models and related to the incidence of disease during the same period. RESULTS An episode of vivax malaria led to 138.6 g (95% confidence interval (CI) 81.9-195.4), 108.6 g (62.8-153.2) and 61 g (20.9-101.1) less weight gain over 2-, 4- and 6-month intervals, respectively. These deficits were larger than both diarrhoea (21.9, 17.2 and 13.8 g less weight gain, respectively) and fever (39.0, 30.3 and 25.6 g less weight gain, respectively). An incident episode of vivax also led to 0.070 cm (0.004-0.137) and 0.083 cm (0.015-0.151) less linear growth over 4 and 6 months, respectively, which were also larger than deficits from diarrhoea (0.029 and 0.028 cm, respectively) and fever (not associated with linear growth deficits). Despite the larger effect of P. vivax incident episodes on growth of a particular child, diarrhoeal disease had a larger cumulative impact on growth deficits as diarrhoeal incidence rates in this community are >10-fold higher than vivax malaria. CONCLUSIONS Disease control measures for vivax malaria and diarrhoeal disease have the potential to improve the growth of children in endemic areas.

Santa Clara de Nanay: The MAL-ED Cohort in Peru

The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study communities in Peru are located in Loreto province, in a rural area 15 km from the city of Iquitos. This riverine population of approximately 5000 individuals is fairly representative of Loreto. The province lags behind the rest of the country in access to water and sanitation, per capita income, and key health indicators including infant mortality (43.0 vs 16.0 per 1000 nationwide) and under-5 mortality (60.6 vs 21.0 per 1000). Total fertility rates are higher than elsewhere in the country (4.3 vs 2.6). Nationwide, the prevalence of human immunodeficiency virus is estimated at 0.45%, the prevalence of tuberculosis is 117 per 100 000, and the incidence of malaria is 258 per 100 000. Stunting in this community is high, whereas acute undernutrition is relatively uncommon. The population suffers from high rates of diarrheal disease. Prevalent enteric pathogens include Ascaris, Giardia, enterotoxigenic Escherichia coli, Shigella, and Campylobacter.

Effects of Shigella-, Campylobacter- and ETEC-associated diarrhea on childhood growth.

Background: Studies examining the etiology-specific effects of diarrheal disease on growth are limited and variable in their analytic methods, making comparisons difficult and priority setting based on these findings challenging. A study by Black et al (Black RE, Brown KH, Becker S. Effects of diarrhea associated with specific enteropathogens on the growth of children in rural Bangladesh. Pediatrics. 1984;33:1004–1009.) examined the association between Shigella and enterotoxigenic Escherichia coli-related disease and weight gain and linear growth in Bangladeshi children aged 0–5 years. We estimated similar associations in a 2002 cohort of 0- to 6-year-old children in the Peruvian Amazon. Methods: Diarrheal surveillence was conducted using household visits 3 times per week. Anthropometry was collected monthly. Mixed-effect models were used to estimate the association between Shigella, ETEC and Campylobacter diarrhea and weight gain in a 2-month period and linear growth over a 9-month period. Diarrheal disease burdens and growth intervals were quantified so as to be as comparable as possible to the original report. Results: Shigella- and ETEC-associated diarrhea were not associated with diminished weight gain, although the association between ETEC diarrhea and weight gain (−4.5 g/percent of days spent with ETEC, P = 0.098) was twice that of other etiologic agents, as well as similar in magnitude to the original report. Shigella-associated diarrhea was associated with decreased linear growth (0.055 cm less growth/percent days, P = 0.008), also similar to the original study. Conclusions: Our findings suggest that associations between enteropathogen-specific diarrheal episodes and growth, particularly Shigella, are comparable across geographic and epidemiological contexts.

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.

Early childhood enteric infections have adverse impacts on child growth and can inhibit normal mucosal responses to oral vaccines, two critical components of environmental enteropathy. To evaluate the role of indoleamine 2,3-dioxygenase 1 (IDO1) activity and its relationship with these outcomes, we measured tryptophan and the kynurenine–tryptophan ratio (KTR) in two longitudinal birth cohorts with a high prevalence of stunting. Children in rural Peru and Tanzania (N = 494) contributed 1,251 plasma samples at 3, 7, 15, and 24 months of age and monthly anthropometrics from 0 to 36 months of age. Tryptophan concentrations were directly associated with linear growth from 1 to 8 months after biomarker assessment. A 1-SD increase in tryptophan concentration was associated with a gain in length-for-age Z-score (LAZ) of 0.17 over the next 6 months in Peru (95% confidence interval [CI] = 0.11–0.23, P < 0.001) and a gain in LAZ of 0.13 Z-scores in Tanzania (95% CI = 0.03–0.22, P = 0.009). Vaccine responsiveness data were available for Peru only. An increase in kynurenine by 1 μM was associated with a 1.63 (95% CI = 1.13–2.34) increase in the odds of failure to poliovirus type 1, but there was no association with tetanus vaccine response. A KTR of 52 was 76% sensitive and 50% specific in predicting failure of response to serotype 1 of the oral polio vaccine. KTR was associated with systemic markers of inflammation, but also interleukin-10, supporting the association between IDO1 activity and immunotolerance. These results strongly suggest that the activity of IDO1 is implicated in the pathophysiology of environmental enteropathy, and demonstrates the utility of tryptophan and kynurenine as biomarkers for this syndrome, particularly in identifying those at risk for hyporesponsivity to oral vaccines.

Lactulose: Mannitol diagnostic test by HPLC and LC-MSMS platforms: Considerations for field studies of intestinal barrier function and environmental enteropathy

Objectives: The lactulose:mannitol (L:M) diagnostic test is frequently used in field studies of environmental enteropathy (EE); however, heterogeneity in test administration and disaccharide measurement has limited the comparison of results between studies and populations. We aim to assess the agreement between L:M measurement between high-performance liquid chromatography with pulsed amperometric detection (HPLC-PAD) and liquid chromatography-tandem mass spectrometry (LC-MSMS) platforms. Methods: The L:M test was administered in a cohort of Peruvian infants considered at risk for EE. A total of 100 samples were tested for lactulose and mannitol at 3 independent laboratories: 1 running an HPLC-PAD platform and 2 running LC-MSMS platforms. Agreement between the platforms was estimated. Results: The Spearman correlation between the 2 LC-MSMS platforms was high (&rgr; ≥ 0.89) for mannitol, lactulose, and the L:M ratio. The correlation between the HPLC-PAD platform and LC-MSMS platform was &rgr; = 0.95 for mannitol, &rgr; = 0.70 for lactulose, and &rgr; = 0.43 for the L:M ratio. In addition, the HPLC-PAD platform overestimated the lowest disaccharide concentrations to the greatest degree. Conclusions: Given the large analyte concentration range, the improved accuracy of LC-MSMS has important consequences for the assessment of lactulose and mannitol following oral administration in populations at risk for EE. We recommend that researchers wishing to implement a dual-sugar test as part of a study of EE use an LC-MSMS platform to optimize the accuracy of results and increase comparability between studies.