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Summary Background Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). Methods GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. Findings We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni or C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2–96·0) at the population level, compared with 51·5% (48·0–55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6–80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. Interpretation A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. Funding Bill & Melinda Gates Foundation.

Background. Diarrhea is a leading cause of illness and death among children aged <5 years in developing countries. This paper describes the clinical and epidemiological methods used to conduct the Global Enteric Multicenter Study (GEMS), a 3-year, prospective, age-stratified, case/control study to estimate the population-based burden, microbiologic etiology, and adverse clinical consequences of acute moderate-to-severe diarrhea (MSD) among a censused population of children aged 0–59 months seeking care at health centers in sub-Saharan Africa and South Asia.Methods. GEMS was conducted at 7 field sites, each serving a population whose demography and healthcare utilization practices for childhood diarrhea were documented. We aimed to enroll 220 MSD cases per year from selected health centers serving each site in each of 3 age strata (0–11, 12–23, and 24–59 months), along with 1–3 matched community controls. Cases and controls supplied clinical, epidemiologic, and anthropometric data at enrollment and again approximately 60 days later, and provided enrollment stool specimens for identification and characterization of potential diarrheal pathogens. Verbal autopsy was performed if a child died. Analytic strategies will calculate the fraction of MSD attributable to each pathogen and the incidence, financial costs, nutritional consequences, and case fatality overall and by pathogen.Conclusions. When completed, GEMS will provide estimates of the incidence, etiology, and outcomes of MSD among infants and young children in sub-Saharan Africa and South Asia. This information can guide development and implementation of public health interventions to diminish morbidity and mortality from diarrheal diseases.

The death of BCG-infected human macrophages induced in vitro by ligation of surface CD95 (Fas), CD69, or complement-mediated lysis was shown not to result in the death of intracellular mycobacteria, whereas exposure to extracellular ATP initiated both macrophage death and killed the intracellular bacteria. ATP acted via P2Z receptors because these effects were mimicked by benzoylbenzoic ATP (a known agonist of P2Z receptors) and blocked by oxidized ATP, DIDS, suramin, amiloride, and KN62 (known inhibitors of P2Z-mediated responses). ATP-mediated bacterial killing was independent of reactive nitrogen and oxygen intermediates and of actinomycin D or cycloheximide inhibition. ATP-induced macrophage cell death, BCG killing, and lucifer yellow dye incorporation were minimal in 2 out of 19 healthy donors. The results suggest possible genetic heterogeneity of this mechanism of mycobacterial killing associated with P2Z-mediated pore formation.

Shigella case isolates from the Global Enteric Multicenter Study were serotyped to guide vaccine development. A quadrivalent vaccine that includes O antigens from S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 should provide broad protection.