Epidemiology of Shigella infections and diarrhea in the first two years of life using culture-independent diagnostics in 8 low-resource settings.

Culture-independent diagnostics have revealed a larger burden of Shigella among children in low-resource settings than previously recognized. We further characterized the epidemiology of Shigella in the first two years of life in a multisite birth cohort. We tested 41,405 diarrheal and monthly non-diarrheal stools from 1,715 children for Shigella by quantitative PCR. To assess risk factors, clinical factors related to age and culture positivity, and associations with inflammatory biomarkers, we used log-binomial regression with generalized estimating equations. The prevalence of Shigella varied from 4.9%-17.8% in non-diarrheal stools across sites, and the incidence of Shigella-attributable diarrhea was 31.8 cases (95% CI: 29.6, 34.2) per 100 child-years. The sensitivity of culture compared to qPCR was 6.6% and increased to 27.8% in Shigella-attributable dysentery. Shigella diarrhea episodes were more likely to be severe and less likely to be culture positive in younger children. Older age (RR: 1.75, 95% CI: 1.70, 1.81 per 6-month increase in age), unimproved sanitation (RR: 1.15, 95% CI: 1.03, 1.29), low maternal education (<10 years, RR: 1.14, 95% CI: 1.03, 1.26), initiating complementary foods before 3 months (RR: 1.10, 95% CI: 1.01, 1.20), and malnutrition (RR: 0.91, 95% CI: 0.88, 0.95 per unit increase in weight-for-age z-score) were risk factors for Shigella. There was a linear dose-response between Shigella quantity and myeloperoxidase concentrations. The burden of Shigella varied widely across sites, but uniformly increased through the second year of life and was associated with intestinal inflammation. Culture missed most clinically relevant cases of severe diarrhea and dysentery.

Enteric dysfunction and other factors associated with attained size at 5 years: MAL-ED birth cohort study findings.

BACKGROUND: Poor growth in early childhood has been associated with increased risk of mortality and morbidity, as well as long-term deficits in cognitive development and economic productivity. OBJECTIVES: Data from the MAL-ED cohort study were used to identify factors in the first 2 y of life that are associated with height-for-age, weight-for-age, and body mass index z-scores (HAZ, WAZ, BMIZ) at 5 y of age. METHODS: A total of 1017 children were followed from near birth until 5 y of age at sites in Bangladesh, Brazil, India, Nepal, Peru, South Africa, and Tanzania. Data were collected on their growth, environmental enteric dysfunction (EED), micronutrient status, enteric pathogen burden, illness prevalence, dietary intake, and various other socio-economic and environmental factors. RESULTS: EED biomarkers were related to size at 5 y. Mean lactulose:mannitol z-scores during the first 2 y of life were negatively associated with all of the growth measures (HAZ: -0.11 [95% CI: -0.19, -0.03]; WAZ: -0.16 [95% CI: -0.26, -0.06]; BMIZ: -0.11 [95% CI: -0.23, 0.0]). Myeloperoxidase was negatively associated with weight (WAZ: -0.52 [95% CI: -0.78, -0.26] and BMIZ: -0.56 [95% CI: -0.86, -0.26]); whereas α-1-antitrypsin had a negative association with HAZ (-0.28 [95% CI: -0.52, -0.04]). Transferrin receptor was positively related to HAZ (0.18 [95% CI: 0.06, 0.30]) and WAZ (0.21 [95% CI: 0.07, 0.35]). Hemoglobin was positively related to HAZ (0.06 [95% CI: 0.00, 0.12]), and ferritin was negatively related to HAZ (-0.08 [95% CI: -0.12, -0.04]). Bacterial density in stool was negatively associated with HAZ (-0.04 [95% CI: -0.08, 0.00]), but illness symptoms did not have any effect on size at 5 y. CONCLUSIONS: EED markers, bacterial density, and iron markers are associated with growth at 5 y of age. Interventions to reduce bacterial burden and EED may improve long-term growth in low-income settings.

Use of quantitative molecular diagnostic methods to investigate the effect of enteropathogen infections on linear growth in children in low-resource settings: longitudinal analysis of results from the MAL-ED cohort study.

BACKGROUND: Enteropathogen infections in early childhood not only cause diarrhoea but contribute to poor growth. We used molecular diagnostics to assess whether particular enteropathogens were associated with linear growth across seven low-resource settings. METHODS: We used quantitative PCR to detect 29 enteropathogens in diarrhoeal and non-diarrhoeal stools collected from children in the first 2 years of life obtained during the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) multisite cohort study. Length was measured monthly. We estimated associations between aetiology-specific diarrhoea and subclinical enteropathogen infection and quantity and attained length in 3 month intervals, at age 2 and 5 years, and used a longitudinal model to account for temporality and time-dependent confounding. FINDINGS: Among 1469 children who completed 2 year follow-up, 35 622 stool samples were tested and yielded valid results. Diarrhoeal episodes attributed to bacteria and parasites, but not viruses, were associated with small decreases in length after 3 months and at age 2 years. Substantial decrements in length at 2 years were associated with subclinical, non-diarrhoeal, infection with Shigella (length-for-age Z score [LAZ] reduction -0·14, 95% CI -0·27 to -0·01), enteroaggregative Escherichia coli (-0·21, -0·37 to -0·05), Campylobacter (-0·17, -0·32 to -0·01), and Giardia (-0·17, -0·30 to -0·05). Norovirus, Cryptosporidium, typical enteropathogenic E coli, and Enterocytozoon bieneusi were also associated with small decrements in LAZ. Shigella and E bieneusi were associated with the largest decreases in LAZ per log increase in quantity per g of stool (-0·13 LAZ, 95% CI -0·22 to -0·03 for Shigella; -0·14, -0·26 to -0·02 for E bieneusi). Based on these models, interventions that successfully decrease exposure to Shigella, enteroaggregative E coli, Campylobacter, and Giardia could increase mean length of children by 0·12-0·37 LAZ (0·4-1·2 cm) at the MAL-ED sites. INTERPRETATION: Subclinical infection and quantity of pathogens, particularly Shigella, enteroaggregative E coli, Campylobacter, and Giardia, had a substantial negative association with linear growth, which was sustained during the first 2 years of life, and in some cases, to 5 years. Successfully reducing exposure to certain pathogens might reduce global stunting. FUNDING: Bill & Melinda Gates Foundation.

Use of quantitative molecular diagnostic methods to assess the aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: a reanalysis of the MAL-ED cohort study.

BACKGROUND: Optimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study. METHODS: We re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0-2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level pathogen-specific attributable burdens, derived stringent quantitative cutoffs to identify aetiology for individual episodes, and created aetiology prediction scores using clinical characteristics. FINDINGS: We analysed 6625 diarrhoeal and 30 968 non-diarrhoeal surveillance stools from 1715 children. Overall, 64·9% of diarrhoea episodes (95% CI 62·6-71·2) could be attributed to an aetiology by quantitative PCR compared with 32·8% (30·8-38·7) using the original study microbiology. Viral diarrhoea (36·4% of overall incidence, 95% CI 33·6-39·5) was more common than bacterial (25·0%, 23·4-28·4) and parasitic diarrhoea (3·5%, 3·0-5·2). Ten pathogens accounted for 95·7% of attributable diarrhoea: Shigella (26·1 attributable episodes per 100 child-years, 95% CI 23·8-29·9), sapovirus (22·8, 18·9-27·5), rotavirus (20·7, 18·8-23·0), adenovirus 40/41 (19·0, 16·8-23·0), enterotoxigenic Escherichia coli (18·8, 16·5-23·8), norovirus (15·4, 13·5-20·1), astrovirus (15·0, 12·0-19·5), Campylobacter jejuni or C coli (12·1, 8·5-17·2), Cryptosporidium (5·8, 4·3-8·3), and typical enteropathogenic E coli (5·4, 2·8-9·3). 86·2% of the attributable incidence for Shigella was non-dysenteric. A prediction score for shigellosis was more accurate (sensitivity 50·4% [95% CI 46·7-54·1], specificity 84·0% [83·0-84·9]) than current guidelines, which recommend treatment only of bloody diarrhoea to cover Shigella (sensitivity 14·5% [95% CI 12·1-17·3], specificity 96·5% [96·0-97·0]). INTERPRETATION: Quantitative molecular diagnostics improved estimates of pathogen-specific burdens of childhood diarrhoea in the community setting. Viral causes predominated, including a substantial burden of sapovirus; however, Shigella had the highest overall burden with a high incidence in the second year of life. These data could improve the management of diarrhoea in these low-resource settings. FUNDING: Bill & Melinda Gates Foundation.

Astrovirus Infection and Diarrhea in 8 Countries.

BACKGROUND AND OBJECTIVES: Astroviruses are important drivers of viral gastroenteritis but remain understudied in community settings and low- and middle-income countries. We present data from 8 countries with high prevalence of diarrhea and undernutrition to describe astrovirus epidemiology and assess evidence for protective immunity among children 0 to 2 years of age. METHODS: We used 25 898 surveillance stools and 7077 diarrheal stools contributed by 2082 children for enteropathogen testing, and longitudinal statistical analysis to describe incidence, risk factors, and protective immunity. RESULTS: Thirty-five percent of children experienced astrovirus infections. Prevalence in diarrheal stools was 5.6%, and severity exceeded all enteropathogens except rotavirus. Incidence of infection and diarrhea were 2.12 and 0.88 episodes per 100 child-months, respectively. Children with astrovirus infection had 2.30 times the odds of experiencing diarrhea after adjustment for covariates (95% confidence interval [CI], 2.01-2.62; P < .001). Undernutrition was a risk factor: odds of infection and diarrhea were reduced by 10% and 13%, respectively, per increase in length-for-age z score (infection: odds ratio, 0.90 [95% CI, 0.85-0.96]; P < .001; diarrhea: odds ratio, 0.87 [95% CI, 0.79-0.96]; P = .006). Some evidence of protective immunity to infection was detected (hazard ratio, 0.84 [95% CI, 0.71-1.00], P = .052), although this was heterogeneous between sites and significant in India and Peru. CONCLUSIONS: Astrovirus is an overlooked cause of diarrhea among vulnerable children worldwide. With the evidence presented here, we highlight the need for future research as well as the potential for astrovirus to be a target for vaccine development.

Age and Sex Normalization of Intestinal Permeability Measures for the Improved Assessment of Enteropathy in Infancy and Early Childhood: Results From the MAL-ED Study.

OBJECTIVES: The aim of the study was to describe changes in intestinal permeability in early childhood in diverse epidemiologic settings. METHODS: In a birth cohort study, the lactulose:mannitol (L:M) test was administered to 1980 children at 4 time points in the first 24 months of life in 8 countries. Data from the Brazil site with an incidence of diarrhea similar to that seen in the United States and no growth faltering was used as an internal study reference to derive age- and sex-specific z scores for mannitol and lactulose recoveries and the L:M ratio. RESULTS: A total of 6602 tests demonstrated mannitol recovery, lactulose recovery, and the L:M ratio were associated with country, sex, and age. There was heterogeneity in the recovery of both probes between sites with mean mannitol recovery ranging for 1.34% to 5.88%, lactulose recovery of 0.19% to 0.58%, and L:M ratios 0.10 to 0.17 in boys of 3 months of age across different sites. We observed strong sex-specific differences in both mannitol and lactulose recovery, with boys having higher recovery of both probes. Alterations in intestinal barrier function increased in most sites from 3 to 9 months of age and plateaued or diminished from 9 to 15 months of age. CONCLUSIONS: Alterations in recovery of the probes differ markedly in different epidemiologic contexts in children living in the developing world. The rate of change in the L:M-z ratio was most rapid and consistently disparate from the reference standard in the period between 6 and 9 months of age, suggesting that this is a critical period of physiologic impact of enteropathy in these populations.

Predicting Undernutrition at Age 2 Years with Early Attained Weight and Length Compared with Weight and Length Velocity.

OBJECTIVE: To estimate the abilities of weight and length velocities vs attained growth measures to predict stunting, wasting, and underweight at age 2 years. STUDY DESIGN: We analyzed data from a community-based cohort study (The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development study [MAL-ED] study) in Bhaktapur, Nepal. A total of 240 randomly selected children were enrolled at birth and followed up monthly up to age 24 months. Linear and logistic regression models were used to predict malnutrition at 2 years of age with growth velocity z scores at 0-3, 0-6, 3-6, 6-9, 6-12, and 9-12 months (using the World Health Organization Growth Standards) or attained growth at 0, 3, 6, and 12 months as predictors. RESULTS: At age 2 years, 4% of the children were wasted, 13% underweight, and 21% stunted. Children who were malnourished at age 2 years had lower mean growth z scores already at birth and throughout the study period. Anthropometric indicators in infancy were significant predictors for growth at the age of 2 years during most periods and at most ages in infancy. Weight-for-age z score, length-for-age z score, and weight-for-length z score at age 12 months had excellent areas under the curve (91-95) to predict the value of the same indicator at age 24 months. Maximum area under the curve values for weight and length velocity were somewhat lower (70-84). CONCLUSIONS: Growth measured at one time point in infancy was better correlated with undernutrition at age 2 years than growth velocity.

Dynamics and Trends in Fecal Biomarkers of Gut Function in Children from 1-24 Months in the MAL-ED Study.

Growth and development shortfalls that are disproportionately prevalent in children living in poor environmental conditions are postulated to result, at least in part, from abnormal gut function. Using data from The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) longitudinal cohort study, we examine biomarkers of gut inflammation and permeability in relation to environmental exposures and feeding practices. Trends in the concentrations of three biomarkers, myeloperoxidase (MPO), neopterin (NEO), and α-1-antitrypsin (AAT), are described from fecal samples collected during the first 2 years of each child's life. A total of 22,846 stool samples were processed during the longitudinal sampling of 2,076 children 0-24 months of age. Linear mixed models were constructed to examine the relationship between biomarker concentrations and recent food intake, symptoms of illness, concurrent enteropathogen infection, and socioeconomic status. Average concentrations of MPO, NEO, and AAT were considerably higher than published references for healthy adults. The concentration of each biomarker tended to decrease over the first 2 years of life and was highly variable between samples from each individual child. Both MPO and AAT were significantly elevated by recent breast milk intake. All three biomarkers were associated with pathogen presence, although the strength and direction varied by pathogen. The interpretation of biomarker concentrations is subject to the context of their collection. Herein, we identify that common factors (age, breast milk, and enteric infection) influence the concentration of these biomarkers. Within the context of low- and middle-income communities, we observe concentrations that indicate gut abnormalities, but more appropriate reference standards are needed.