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.

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.

Etiology of Severe Acute Watery Diarrhea in Children in the Global Rotavirus Surveillance Network Using Quantitative Polymerase Chain Reaction.

Background: The etiology of acute watery diarrhea remains poorly characterized, particularly after rotavirus vaccine introduction. Methods: We performed quantitative polymerase chain reaction for multiple enteropathogens on 878 acute watery diarrheal stools sampled from 14643 episodes captured by surveillance of children <5 years of age during 2013-2014 from 16 countries. We used previously developed models of the association between pathogen quantity and diarrhea to calculate pathogen-specific weighted attributable fractions (AFs). Results: Rotavirus remained the leading etiology (overall weighted AF, 40.3% [95% confidence interval {CI}, 37.6%-44.3%]), though the AF was substantially lower in the Americas (AF, 12.2 [95% CI, 8.9-15.6]), based on samples from a country with universal rotavirus vaccination. Norovirus GII (AF, 6.2 [95% CI, 2.8-9.2]), Cryptosporidium (AF, 5.8 [95% CI, 4.0-7.6]), Shigella (AF, 4.7 [95% CI, 2.8-6.9]), heat-stable enterotoxin-producing Escherichia coli (ST-ETEC) (AF, 4.2 [95% CI, 2.0-6.1]), and adenovirus 40/41 (AF, 4.2 [95% CI, 2.9-5.5]) were also important. In the Africa Region, the rotavirus AF declined from 54.8% (95% CI, 48.3%-61.5%) in rotavirus vaccine age-ineligible children to 20.0% (95% CI, 12.4%-30.4%) in age-eligible children. Conclusions: Rotavirus remained the leading etiology of acute watery diarrhea despite a clear impact of rotavirus vaccine introduction. Norovirus GII, Cryptosporidium, Shigella, ST-ETEC, and adenovirus 40/41 were also important. Prospective surveillance can help identify priorities for further reducing the burden of diarrhea.