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.

HighlightsOral vaccines exhibit poor performance in low-income settings.Enterovirus and Campylobacter carriage are associated with lower OPV immunogenicity.Enterovirus carriage is associated with lower Rotarix immunogenicity and efficacy.

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1β, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

Childhood diarrhea in low-resource settings has been variably linked to linear growth shortfalls. However, the association between etiology-specific diarrhea and growth has not been comprehensively evaluated. We tested diarrheal stools collected from the Performance of Rotavirus and Oral Polio Vaccines in Developing Countries study from 2011 to 2013 in Dhaka, Bangladesh, by quantitative polymerase chain reaction for a broad range of enteropathogens to characterize diarrhea etiology and examine the association between etiology-specific diarrhea and linear growth and systemic inflammation. Pathogen-specific burdens of diarrhea were determined using attributable fractions. Linear regression was used to examine associations of pathogen-specific diarrhea with length-for-age z scores (LAZ) and serum C-reactive protein. There was no relationship between all-cause diarrhea and length at 12 months (change in 12-month LAZ per episode, −0.01, 95% confidence interval (CI): −0.06, 0.03). However, Cryptosporidium (change in 12-month LAZ per attributable episode, −0.23, 95% CI: −0.50, 0.03), Campylobacter jejuni/coli (change of −0.16, 95% CI: −0.32, −0.01), and Shigella/enteroinvasive Escherichia coli diarrhea (change of −0.12, 95% CI: −0.26, 0.03) were associated with linear growth deficits. Diarrhea attributable to C. jejuni/coli and Shigella/enteroinvasive E. coli were associated with elevated C-reactive protein. The association between diarrhea and linear growth appears to be pathogen-specific, reinforcing the need for pathogen-specific interventions.

SummaryBackgroundTrivalent oral polio vaccine (tOPV) was replaced worldwide from April, 2016, by bivalent types 1 and 3 oral polio vaccine (bOPV) and one dose of inactivated polio vaccine (IPV) where available. The risk of transmission of type 2 poliovirus or Sabin 2 virus on re-introduction or resurgence of type 2 poliovirus after this switch is not understood completely. We aimed to assess the risk of Sabin 2 transmission after a polio vaccination campaign with a monovalent type 2 oral polio vaccine (mOPV2).MethodsWe did an open-label cluster-randomised trial in villages in the Matlab region of Bangladesh. We randomly allocated villages (clusters) to either: tOPV at age 6 weeks, 10 weeks, and 14 weeks; or bOPV at age 6 weeks, 10 weeks, and 14 weeks and either one dose of IPV at age 14 weeks or two doses of IPV at age 14 weeks and 18 weeks. After completion of enrolment, we implemented an mOPV2 vaccination campaign that targeted 40% of children younger than 5 years, regardless of enrolment status. The primary outcome was Sabin 2 incidence in the 10 weeks after the campaign in per-protocol infants who did not receive mOPV2, as assessed by faecal shedding of Sabin 2 by reverse transcriptase quantitative PCR (RT-qPCR). The effect of previous immunity on incidence was also investigated with a dynamical model of poliovirus transmission to observe prevalence and incidence of Sabin 2 virus. This trial is registered at ClinicalTrials.gov, number NCT02477046.FindingsBetween April 30, 2015, and Jan 14, 2016, individuals from 67 villages were enrolled to the study. 22 villages (300 infants) were randomly assigned tOPV, 23 villages (310 infants) were allocated bOPV and one dose of IPV, and 22 villages (329 infants) were assigned bOPV and two doses of IPV. Faecal shedding of Sabin 2 in infants who did not receive the mOPV2 challenge did not differ between children immunised with bOPV and one or two doses of IPV and those who received tOPV (15 of 252 [6%] vs six of 122 [4%]; odds ratio [OR] 1·29, 95% CI 0·45–3·72; p=0·310). However, faecal shedding of Sabin 2 in household contacts was increased significantly with bOPV and one or two doses of IPV compared with tOPV (17 of 751 [2%] vs three of 353 [1%]; OR 3·60, 95% CI 0·82–15·9; p=0·045). Dynamical modelling of within-household incidence showed that immunity in household contacts limited transmission.InterpretationIn this study, simulating 1 year of tOPV cessation, Sabin 2 transmission was higher in household contacts of mOPV2 recipients in villages receiving bOPV and either one or two doses of IPV, but transmission was not increased in the community as a whole as shown by the non-significant difference in incidence among infants. Dynamical modelling indicates that transmission risk will be higher with more time since cessation.FundingBill & Melinda Gates Foundation.

The study assessed the impact of an EPI (Expanded Programme on Immunization) intervention package, implemented within the existing service-delivery system, to improve the child immunization coverage in urban slums of Dhaka, Bangladesh. This intervention trial used a pre- and post-test design. An intervention package was tested from September 2006 to August 2007 in two urban slums. The intervention package included: (a) an extended EPI service schedule; (b) training for service providers on valid doses and management of side-effects; (c) a screening tool to identify immunization needs among clinic attendants; and (d) an EPI support group for social mobilization. Data were obtained from random sample surveys, service statistics and qualitative interviews. Analysis of quantitative data was based on a 'before and after' assessment of selected immunization-coverage indicators. Qualitative data were analysed using content analysis. Ninety-nine per cent of the children were fully immunized after implementation of the interventions compared with only 43% before implementation. Antigen-wise coverage after implementation was also significantly higher compared with before implementation. Only 1% drop-out was observed after implementation of the interventions while it was 33% before implementation. At baseline, a significantly higher proportion of children of non-working mothers (75%) were fully immunized compared with children of working mothers (14%). Although the proportion of fully immunized children of both non-working and working mothers was significantly higher at endline, fully immunized children of working mothers dramatically improved at endline (99%) compared with baseline (14%). The findings suggest the effectiveness of a 'package of interventions' in improving child immunization coverage in urban slums. However, further research is needed to fully assess the effectiveness of the package, to assess the individual components in order to identify those that make the biggest contribution to coverage, and to assess the sustainability of this package within the existing service delivery system, particularly on a wider scale.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C . scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E . histolytica . Introduction of C . scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C . scindens –colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E . histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C . scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C . scindens –colonized specific pathogen–free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C . scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E . histolytica .

Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], ؉4, ؉11, ؉18, and ؉25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture ؉ / qPCR ؉ and culture ؊ /qPCR ؉ stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day ؉11 or ؉18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. Both immunity to paralytic poliomyelitis and reduction of transmission of wild-type and vaccine-derived polioviruses are critical in polio eradication efforts (1, 2). Oral polio vaccine (OPV) is widely utilized in low-income settings for ease of delivery, low cost, and potential for mucosal immunity. OPV immunogenicity is imperfect (3), however, and shedding of vaccine strains further complicates the poliovirus reservoir. Measurement of poliovirus in stool samples can thus be useful in evaluating mucosal immunogenicity (4), in estimating the transmission intensities of vaccine-derived virus, and for surveillance after polio eradication (5).Poliovirus culture is still considered the gold standard procedure but requires up to two 7-day passages in two cell lines, followed by molecular or antigenic methods to differentiate and type the poliovirus isolates (6). This process is time-consuming and greatly diminishes the number of stool samples that can be surveyed. Furthermore, subcultured isolates are not always representative of the viruses present in the original stool sample and results are usually not quantitated by titer determination (7,8). PCR methods are therefore attractive in terms of cost, time, the ability to subtype, and quantitation (9-12). PCR for poliovirus has been found to be severalfold more sensitive than poliovirus isolation from cell culture (13) and has also been found to be more sensitive with cerebrospinal fluid, throat swabs, or stool samples (14-17). However, s...