Global Enteric Multicenter Study Research Articles

Association between Pathogenic Variants of Diarrheagenic Escherichia coli and Growth in Children under 5 Years of Age in the Global Enteric Multicenter Study.

ABSTRACT.There is a lack of information highlighting associations between different pathogenic variants of diarrheagenic Escherichia coli and childhood growth. Pathogenic variants of E. coli from stool samples, collected from 22,567 children enrolled in the Global Enteric Multicenter Study from December 2007 to March 2011, were detected by real-time polymerase chain reaction. We estimated the associations of different pathogenic variants of diarrheagenic E. coli with child growth. The association between an explanatory variable and the outcome variable was assessed using multiple linear regression, where the dependent variables were height-for-age, weight-for-age, and weight-for-height z-scores, and the independent variable was the presence of different pathogenic variants of diarrheagenic E. coli. After adjusting for potential covariates, such as age, gender, diarrhea, breastfeeding status, mother’s education, number of under-5 children, handwashing practice, handwashing material, source of drinking water, wealth index, available toilet facility, copathogens, comorbidity, time, and study site, the multivariable model identified a negative association between different pathogenic variants of diarrheagenic E. coli and child growth. Our analyses may provide the cornerstone for prospective epidemiologic investigation for the development of preventive measures for diarrheagenic E. coli and combat childhood undernutrition.

Pathogenomic analyses of Shigella isolates inform factors limiting shigellosis prevention and control across LMICs

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMICs), are increasingly antimicrobial resistant and have no widely available licenced vaccine. We performed genomic analyses of 1,246 systematically collected shigellae sampled from seven countries in sub-Saharan Africa and South Asia as part of the Global Enteric Multicenter Study (GEMS) between 2007 and 2011, to inform control and identify factors that could limit the effectiveness of current approaches. Through contemporaneous comparison among major subgroups, we found that S. sonnei contributes ≥6-fold more disease than other Shigella species relative to its genomic diversity, and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against ciprofloxacin, the current WHO-recommended antimicrobial for the treatment of shigellosis, among Shigella isolates. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, providing a framework for the focused application of comparative genomics to guide vaccine development, and the optimization of control and prevention strategies for other pathogens relevant to public health policy considerations.

Anthropometric Indices of Giardia-Infected Under-Five Children Presenting with Moderate-to-Severe Diarrhea and Their Healthy Community Controls: Data from the Global Enteric Multicenter Study.

Among all intestinal parasitosis, giardiasis has been reported to be associated with delayed growth in malnourished children under 5 living in low- and middle-income countries. Relevant data on the nutritional status of children aged 0–59 months presenting with moderate-to-severe diarrhea (MSD) and giardia infection were collected from sentinel health facilities of the Global Enteric Multicenter Study’s (GEMS) seven field settings, placed in diverse countries of Sub-Saharan Africa and South Asia between, December 2007 and February 2011. Then, this study analyzed a robust dataset of study participants (n = 22,569). Children having giardiasis with MSD constituted as cases (n = 1786), and those without MSD constituted as controls (n = 3470). Among the seven field sites, symptomatic giardiasis was 15% and 22% in Asian and African sites, respectively, whereas asymptomatic giardia infection (healthy without MSD) in Asian and African sites was 21.7% and 30.7%, respectively. Wasting and underweight were more frequently associated and stunting less often associated with symptomatic giardiasis (for all, p < 0.001). Symptomatic giardiasis had a significant association with worsening of nutritional status in under-five children. Improved socio-economic profile along with proper sanitation and hygienic practices are imperative to enhance child nutritional status, particularly in resource limited settings.

Nontyphoidal Salmonella among Children under 5 Years Old in Sub-Saharan Africa and South Asia in the Global Enteric Multicenter Study.

ABSTRACT.Factors associated with nontyphoidal Salmonella (NTS) infection have not been well characterized to date. We aimed to compare the associated factors among children under age 5 years with NTS infection in sub-Saharan Africa and South Asia. Data from children having moderate-to-severe diarrhea (MSD) and asymptomatic children with NTS isolated from fecal specimens were extracted from the Global Enteric Multicenter Study (GEMS), conducted from December 2007 to March 2011. Compared with NTS-negative children, NTS-associated MSD cases in South Asia were associated with the presence of goat in the house (adjusted odds ratio [aOR]: 2.15; 95% confidence interval [CI]: 1.25–3.70) and handwashing after handling an animal (aOR: 2.26; 95% CI: 1.36–3.74). In sub-Saharan Africa, children with NTS associated MSD had a greater association with stunting (1.21 95% CI: 1.01–1.45), longer duration of diarrhea (aOR: 1.25 95% CI: 1.19–1.31); presence of cow in house (aOR: 1.54 95% CI: 1.09–2.16), handwashing after handling animal (aOR: 2.41 95% CI: 1.74–3.33). Drinking tube well water (aOR: 0.54 95% CI: 0.32–0.91), availability of toilet facility (aOR: 0.58 95% CI: 0.53–0.65), and handwashing before eating (aOR: 0.76 95% CI: 0.57–1.00) and after defecation (aOR: 0.80 95% CI: 0.69, 0.94) were found to be protective. The differentials between children of both regions having fecal NTS are distinct and underscore the need for policymaking for preventive and control strategies targeting stunted children.

THE TALE OF THE TRAVELING CHEESE: SHIGELLA IN A LUNG TRANSPLANT PATIENT

TOPIC: Transplantation TYPE: Fellow Case Reports INTRODUCTION: Shigella is a common cause of gastroenteritis in areas of overcrowding and poor sanitation, but is seen less frequently in the developed world. Infection is mainly acquired through the fecal-oral route, but consumption of unpasteurized dairy remains a high risk for transmission. In this report, we describe a case of a lung transplant patient who presented with septic shock secondary to Shigella gastroenteritis after ingesting unpasteurized cheese brought back from Peru. CASE PRESENTATION: This is a case of a 62-year-old Peruvian man who underwent a bilateral lung transplant for end stage idiopathic pulmonary fibrosis in November 2018, a year prior to this presentation. Pre-transplant the patient status was CMV +/+, EBV +/+. At time of presentation his immunosuppression regimen consisted of Tacrolimus 0.5 mg, Mycophenolate 500mg twice daily and Prednisone 10mg daily.He had been progressing well until he presented to the emergency department with a reported fever of 103 degrees Fahrenheit, around 40 episodes a day of profuse non-bloody, watery diarrhea and abdominal discomfort. He did not complain of nausea or vomiting. He revealed that he had eaten soft, white, unpasteurized cheese that was brought from Peru two days prior.He was hypotensive with a blood pressure of 72/39, a heart rate of 107 beats per minute and SaO2 of 96% while breathing ambient air. A physical exam revealed mild diffuse abdominal tenderness, rest of exam was normal.Laboratory examination revealed a white blood cell count of 18,000 cells/mcl, platelet (PLT) count of 147,000 cells/mcl, creatinine 1.30 mg/dL, lactic acid 3.2 mmol/L. Computerized tomography (CT) of his abdomen showed fluid filled loops of the colon and small bowel, reflecting enteritis and diarrhea. Stool studies were sent. The patient was treated with intravenous fluids, Tacrolimus was held due to new acute kidney injury, Mycophenolate held due to leukopenia and Cefepime and Metronidazole were administered for empiric coverage for abdominal pathogens. On day 4 of admission Shigella flexneri (sensitive to Levofloxacin and Ceftriaxone, resistant to Ampicillin) was isolated and infectious disease consultation obtained. The patient had clinically recovered on Cefepime and he completed the antibiotic course. He was discharged the following day on his routine triple immunosuppressive therapy. DISCUSSION: Solid organ transplant recipients with diarrhea should be tested for shigella. Patients not responding to conventional treatment should undergo repeat culture and sensitivity testing. Bacterial culture is the gold standard for diagnosis of shigella infections. The use of antibiotics depends on the severity of illness, the age of the patient and immune status. CONCLUSIONS: This case highlights the importance of educating transplant patients on how to reduce certain harmful exposures that may be fatal in immunosuppressed individuals. REFERENCE #1: 1. GBD 2016 Diarrhoeal Disease Collaborators. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of diarrhoea in 195 countries: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Infect Dis 2018. REFERENCE #2: 2. Kotloff KL, Winickoff JP, Ivanoff B, et al. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull World Health Organ 1999; 77:651–66 REFERENCE #3: 3. Livio S, Strockbine NA, Panchalingam S, et al. Shigella isolates from the global enteric multicenter study inform vaccine development. Clin Infect Dis 2014; 59: 933–41. DISCLOSURES: No relevant relationships by Nikhil Madan, source=Web Response No relevant relationships by Vipul Patel, source=Web Response No relevant relationships by Safiyya Quintiliani, source=Web Response

Pathogens Associated With Linear Growth Faltering in Children With Diarrhea and Impact of Antibiotic Treatment: The Global Enteric Multicenter Study.

BackgroundThe association between childhood diarrheal disease and linear growth faltering in developing countries is well described. However, the impact attributed to specific pathogens has not been elucidated, nor has the impact of recommended antibiotic treatment.MethodsThe Global Enteric Multicenter Study enrolled children with moderate to severe diarrhea (MSD) seeking healthcare at 7 sites in sub-Saharan Africa and South Asia. At enrollment, we collected stool samples to identify enteropathogens. Length/height was measured at enrollment and follow-up, approximately 60 days later, to calculate change in height-for-age z scores (ΔHAZ). The association of pathogens with ΔHAZ was tested using linear mixed effects regression models.ResultsAmong 8077 MSD cases analyzed, the proportion with stunting (HAZ below −1) increased from 59% at enrollment to 65% at follow-up (P < .0001). Pathogens significantly associated with linear growth decline included Cryptosporidium (P < .001), typical enteropathogenic Escherichia coli (P = .01), and untreated Shigella (P = .009) among infants (aged 0–11 months) and enterotoxigenic E. coli encoding heat-stable toxin (P < .001) and Cryptosporidium (P = .03) among toddlers (aged 12–23 months). Shigella-infected toddlers given antibiotics had improved linear growth (P = .02).ConclusionsLinear growth faltering among children aged 0–23 months with MSD is associated with specific pathogens and can be mitigated with targeted treatment strategies, as demonstrated for Shigella.

Association of enteropathogen detection with diarrhoea by age and high versus low child mortality settings: a systematic review and meta-analysis

SummaryBackgroundThe odds ratio (OR) comparing pathogen presence in diarrhoeal cases versus asymptomatic controls is a measure for diarrhoeal disease cause that has been integrated into burden of disease estimates across diverse populations. This study aimed to estimate the OR describing the association between pathogen detection in stool and diarrhoea for 15 common enteropathogens by age group and child mortality setting.MethodsWe did a systematic review to identify case-control and cohort studies published from Jan 1, 1990, to July 9, 2019, which examined at least one enteropathogen of interest and the outcome diarrhoea. The analytical dataset included data extracted from published articles and supplemented with data from the Global Enteric Multicenter Study and the Malnutrition and Enteric Disease study. Random effects meta-analysis models were fit for each enteropathogen, stratified by age group and child mortality level, and adjusted for pathogen detection method and study design to produce summary ORs describing the association between pathogen detection in stool and diarrhoea.Findings1964 records were screened and 130 studies (over 88 079 cases or diarrhoea samples and 135 755 controls or non-diarrhoea samples) were available for analysis. Heterogeneity (I2) in unadjusted models was substantial, ranging from 27·6% to 86·6% across pathogens. In stratified and adjusted models, summary ORs varied by age group and setting, ranging from 0·4 (95% CI 0·2–0·6) for Giardia lamblia to 54·1 (95% CI 7·4–393·5) for Vibrio cholerae.InterpretationIncorporating effect estimates from diverse data sources into diarrhoeal disease cause and burden of disease models is needed to produce more representative estimates.FundingWHO, Bill & Melinda Gates Foundation, and National Institutes of Health.

Associated factors, post infection child growth, and household cost of invasive enteritis among under 5 children in Bangladesh

Both Campylobacter- and Shigella-induced invasive enteritis are common in under-5 Bangladeshi children. Our study aimed to determine the factors associated with Campylobacter and Shigella enteritis among under-5 children, the post-infection worsening growth, and the household cost of invasive enteritis. Data of children having Shigella (591/803) and Campylobacter (246/1148) isolated from the fecal specimen in Bangladesh were extracted from the Global Enteric Multicenter Study (GEMS) for the period December 2007 to March 2011. In multiple logistic regression analysis, fever was observed more frequently among shigellosis cases [adjusted OR 2.21; (95% CI 1.58, 3.09)]. Breastfeeding [aOR 0.55; (95% CI 0.37, 0.81)] was found to be protective against Shigella. The generalized estimating equations multivariable model identified a negative association between Shigella and weight-for-height z score [aOR − 0.11; (95% CI − 0.21, − 0.001)]; a positive association between symptomatic Campylobacter and weight-for-age z score [aOR 0.22; (95% CI 0.06, 0.37)] and weight-for-height z score [aOR 0.22; (95% CI 0.08, 0.37)]. Total costs incurred by households were more in shigellosis children than Campylobacter-induced enteritis ($4.27 vs. $3.49). Households with low-level maternal education tended to incur less cost in case of their shigellosis children. Our findings underscore the need for preventive strategies targeting Shigella infection, which could potentially reduce the disease burden, associated household costs, and child growth faltering.

Molecular Characterisation of Cryptosporidium spp. in Mozambican Children Younger than 5 Years Enrolled in a Matched Case-Control Study on the Aetiology of Diarrhoeal Disease.

Cryptosporidium is a leading cause of childhood diarrhoea and associated physical and cognitive impairment in low-resource settings. Cryptosporidium-positive faecal samples (n = 190) from children aged ≤ 5 years enrolled in the Global Enteric Multicenter Study (GEMS) in Mozambique detected by ELISA (11.5%, 430/3754) were successfully PCR-amplified and sequenced at the gp60 or ssu rRNA loci for species determination and genotyping. Three Cryptosporidium species including C. hominis (72.6%, 138/190), C. parvum (22.6%, 43/190), and C. meleagridis (4.2%, 8/190) were detected. Children ≤ 23 months were more exposed to Cryptosporidium spp. infections than older children. Both C. hominis and C. parvum were more prevalent among children with diarrhoeal disease compared to those children without it (47.6% vs. 33.3%, p = 0.007 and 23.7% vs. 11.8%, p = 0.014, respectively). A high intra-species genetic variability was observed within C. hominis (subtype families Ia, Ib, Id, Ie, and If) and C. parvum (subtype families IIb, IIc, IIe, and IIi) but not within C. meleagridis (subtype family IIIb). No association between Cryptosporidium species/genotypes and child’s age was demonstrated. The predominance of C. hominis and C. parvum IIc suggests that most of the Cryptosporidium infections were anthroponotically transmitted, although zoonotic transmission events also occurred at an unknown rate. The role of livestock, poultry, and other domestic animal species as sources of environmental contamination and human cryptosporidiosis should be investigated in further molecular epidemiological studies in Mozambique.

Rotavirus disease burden pre-vaccine introduction in young children in Rural Southern Mozambique, an area of high HIV prevalence.

BackgroundRotavirus vaccines have been adopted in African countries since 2009, including Mozambique (2015). Disease burden data are needed to evaluate the impact of rotavirus vaccine. We report the burden of rotavirus-associated diarrhea in Mozambique from the Global Enteric Multicenter Study (GEMS) before vaccine introduction.MethodsA case-control study (GEMS), was conducted in Manhiça district, recruiting children aged 0–59 months with moderate-to-severe diarrhea (MSD) and less-severe-diarrhea (LSD) between December 2007 and November 2012; including 1–3 matched (age, sex and neighborhood) healthy community controls. Clinical and epidemiological data and stool samples (for laboratory investigation) were collected. Association of rotavirus with MSD or LSD was determined by conditional logistic regression and adjusted attributable fractions (AF) calculated, and risk factors for rotavirus diarrhea assessed.ResultsOverall 915 cases and 1,977 controls for MSD, and 431 cases and 430 controls for LSD were enrolled. Rotavirus positivity was 44% (217/495) for cases and 15% (160/1046) of controls, with AF = 34.9% (95% CI: 32.85–37.06) and adjusted Odds Ratio (aOR) of 6.4 p< 0.0001 in infants with MSD compared to 30% (46/155) in cases and 14% (22/154) in controls yielding AF = 18.7%, (95% CI: 12.02–25.39) and aOR = 2.8, p = 0.0011 in infants with LSD. The proportion of children with rotavirus was 32% (21/66) among HIV-positive children and 23% (128/566) among HIV-negative ones for MSD. Presence of animals in the compound (OR = 1.9; p = 0.0151) and giving stored water to the child (OR = 2.0, p = 0.0483) were risk factors for MSD; while animals in the compound (OR = 2.37, p = 0.007); not having routine access to water on a daily basis (OR = 1.53, p = 0.015) and washing hands before cooking (OR = 1.76, p = 0.0197) were risk factors for LSD.ConclusionThe implementation of vaccination against rotavirus may likely result in a significant reduction of rotavirus-associated diarrhea, suggesting the need for monitoring of vaccine impact.

Target Product Profile and Development Path for Shigellosis Treatment with Antibacterials.

Enteric infection with Shigella spp. can lead to symptoms ranging from acute watery diarrhea to sudden, severe dysentery. Approximately 212 000 diarrheal deaths annually are attributed to Shigella with a disproportionate impact in low-resource countries. The impact in under-resourced countries was illustrated by a reanalysis of the Global Enteric Multicenter Study which found that Shigella was the leading pathogen associated with moderate-to severe diarrhea in children under 5 years old. While recent studies have highlighted the burden of the disease, there has been a concurrent reduction in therapeutic options for the treatment of shigellosis as drug resistant strains increase in prevalence. In addition, increasing reports of drug resistant shigellosis cases in the men who have sex with men community confirm that the impact is not limited to low-resource countries. Despite the urgent need for new treatments, a target product profile (TPP) has not been established, and there is no clear development path for antibacterial treatments. To address this troubling concern, this manuscript describes a TPP for antishigellosis small molecule therapeutics and a development path that integrates currently available preclinical and clinical models of Shigella infection.

Association between Non-Typhoidal Salmonella Infection and Growth in Children under 5 Years of Age: Analyzing Data from the Global Enteric Multicenter Study.

Non-typhoidal Salmonella (NTS) is one of the less focused on infections and is often associated with faulty child nutrition in the developing world. This study aimed to evaluate the association of NTS infection with growth faltering among children under the age of five. We analyzed data from 378 fecal NTS positive children with both moderate-to-severe diarrhea (MSD) and asymptomatic infection from the seven countries of South Asia and sub-Saharan Africa during enrolment and on day 60 follow up in the Global Enteric Multicenter Study (GEMS) for the period of December 2007 to March 2011. Children not associated with fecal NTS (n = 1134) were randomly selected from the same dataset (1:3 ratio) as a comparison group. The association between an explanatory variable and the outcome variable was longitudinally tested using generalized estimating equations (GEE), where the dependent variables were height-for-age (HAZ), weight-for-age (WAZ), and weight-for-height (WHZ) z-score, and the independent variable was the presence of fecal NTS. The GEE multivariable model identified a negative association between fecal NTS and WAZ (coefficient: −0.19; 95% CI (confidence interval): −0.33, −0.04, and p value = 0.010), WHZ (coef: −0.19; 95% CI: −0.34, −0.05, and p value = 0.007), and HAZ (coef: −0.13; 95% CI: −0.27, −0.01, and p value = 0.073) after adjusting for age, gender, diarrhea, breastfeeding status, mothers’ education, number of children under the age of five, household size by the number of people regularly sleep at the home, handwashing practice, source of drinking water, wealth index, presence of co-pathogens, comorbidity, and study sites. In the GEMS, where children were followed during 50–90 days of enrolment, the presence of fecal NTS harmed the child’s anthropometric outcomes. Minimizing potential exposure to NTS is needed to curb worsening child undernutrition.

Characteristics of Salmonella Recovered From Stools of Children Enrolled in the Global Enteric Multicenter Study.

BackgroundThe Global Enteric Multicenter Study (GEMS) determined the etiologic agents of moderate-to-severe diarrhea (MSD) in children under 5 years old in Africa and Asia. Here, we describe the prevalence and antimicrobial susceptibility of nontyphoidal Salmonella (NTS) serovars in GEMS and examine the phylogenetics of Salmonella Typhimurium ST313 isolates.MethodsSalmonella isolated from children with MSD or diarrhea-free controls were identified by classical clinical microbiology and serotyped using antisera and/or whole-genome sequence data. We evaluated antimicrobial susceptibility using the Kirby-Bauer disk-diffusion method. Salmonella Typhimurium sequence types were determined using multi-locus sequence typing, and whole-genome sequencing was performed to assess the phylogeny of ST313.ResultsOf 370 Salmonella-positive individuals, 190 (51.4%) were MSD cases and 180 (48.6%) were diarrhea-free controls. The most frequent Salmonella serovars identified were Salmonella Typhimurium, serogroup O:8 (C2-C3), serogroup O:6,7 (C1), Salmonella Paratyphi B Java, and serogroup O:4 (B). The prevalence of NTS was low but similar across sites, regardless of age, and was similar among both cases and controls except in Kenya, where Salmonella Typhimurium was more commonly associated with cases than controls. Phylogenetic analysis showed that these Salmonella Typhimurium isolates, all ST313, were highly genetically related to isolates from controls. Generally, Salmonella isolates from Asia were resistant to ciprofloxacin and ceftriaxone, but African isolates were susceptible to these antibiotics.ConclusionsOur data confirm that NTS is prevalent, albeit at low levels, in Africa and South Asia. Our findings provide further evidence that multidrug-resistant Salmonella Typhimurium ST313 can be carried asymptomatically by humans in sub-Saharan Africa.

Molecular diversity of Giardia duodenalis in children under 5 years from the Manhiça district, Southern Mozambique enrolled in a matched case-control study on the aetiology of diarrhoea.

Giardia duodenalis is an enteric parasite commonly detected in children. Exposure to this organism may lead to asymptomatic or symptomatic infection. Additionally, early-life infections by this protozoan have been associated with impaired growth and cognitive function in poor resource settings. The Global Enteric Multicenter Study (GEMS) in Mozambique demonstrated that G. duodenalis was more frequent among controls than in diarrhoeal cases (≥3 loosing stools in the previous 24 hours). However, no molecular investigation was conducted to ascertain the molecular variability of the parasite. Therefore, we describe here the frequency and genetic diversity of G. duodenalis infections in children younger than five years of age with and without diarrhoea from the Manhiça district in southern Mozambique enrolled in the context of GEMS. Genomic DNA from 757 G. duodenalis-positive stool samples by immunoassay collected between 2007-2012, were reanalysed by multiplex PCR targeting the E1-HP and C1-P21 genes for the differentiation of assemblages A and B. Overall, 47% (353) of the samples were successfully amplified in at least one locus. Assemblage B accounted for 90% (319/353) of all positives, followed by assemblage A (8%, 29/353) and mixed A+B infections (1%, 5/353). No association between the presence of a given assemblage and the occurrence of diarrhoea could be demonstrated. A total of 351 samples were further analysed by a multi-locus sequence genotyping (MLSG) approach at the glutamate dehydrogenase (gdh), ß-giardin (bg) and triose phosphate isomerase (tpi) genes. Overall, 63% (222/351) of samples were genotyped and/or sub-genotyped in at least one of the three markers. Sequence analysis revealed the presence of assemblages A (10%; 23/222) and B (90%; 199/222) with high molecular diversity at the nucleotide level within the latter; no mixed infections were identified under the MLSG scheme. Assemblage A sequences were assigned to sub-assemblages AI (0.5%, 1/222), AII (7%, 15/222) or ambiguous AII/AIII (3%, 7/222). Within assemblage B, sequences were assigned to sub-assemblages BIII (13%, 28/222), BIV (14%, 31/222) and ambiguous BIII/BIV (59%, 132/222). BIII/BIV sequences accumulated the majority of the single nucleotide polymorphisms detected, particularly in the form of double peaks at chromatogram inspection. This study demonstrated that the occurrence of gastrointestinal illness (diarrhoea) was not associated to a given genotype of G. duodenalis in Mozambican children younger than five years of age. The assemblage B of the parasite was responsible for nine out of ten infections detected in this paediatric population. The extremely high genetic diversity observed within assemblage B isolates was compatible with an hyperendemic epidemiological scenario where infections and reinfections were common. The obtained molecular data may be indicative of high coinfection rates by different G. duodenalis assemblages/sub-assemblages and/or genetic recombination events, although the exact contribution of both mechanisms to the genetic diversity of the parasite remains unknown.

The Clinical Presentation of Culture-positive and Culture-negative, Quantitative Polymerase Chain Reaction (qPCR)-Attributable Shigellosis in the Global Enteric Multicenter Study and Derivation of a Shigella Severity Score: Implications for Pediatric Shigella Vaccine Trials

Background Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials.MethodsWe compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score.ResultsCompared to culture-positive Shigella MSD cases (n = 745), culture-negative/qPCR-attributable Shigella cases (n = 852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There was no difference in dehydration, hospitalization, or severe classification from a modified Vesikari score. Twenty-two (1.8%) Shigella MSD cases died within the 14-days after presentation to health facilities, and 59.1% of these deaths were in culture-negative cases. Age <12 months, diarrhea duration prior to presentation, vomiting, stunting, wasting, and hospitalization were associated with mortality. A model-derived score assigned points for dehydration, hospital admission, and longer diarrhea duration but was not significantly better at predicting 14-day mortality than a modified Vesikari score.ConclusionsA composite severity score consistent with severe disease or dysentery may be a pragmatic clinical endpoint for severe shigellosis in vaccine trials. Reliance on culture for microbiologic confirmation may miss a substantial number of Shigella cases but is currently required to measure serotype specific immunity.

Clinical predictors for etiology of acute diarrhea in children in resource-limited settings.

BackgroundDiarrhea is one of the leading causes of childhood morbidity and mortality in lower- and middle-income countries. In such settings, access to laboratory diagnostics are often limited, and decisions for use of antimicrobials often empiric. Clinical predictors are a potential non-laboratory method to more accurately assess diarrheal etiology, the knowledge of which could improve management of pediatric diarrhea.MethodsWe used clinical and quantitative molecular etiologic data from the Global Enteric Multicenter Study (GEMS), a prospective, case-control study, to develop predictive models for the etiology of diarrhea. Using random forests, we screened the available variables and then assessed the performance of predictions from random forest regression models and logistic regression models using 5-fold cross-validation.ResultsWe identified 1049 cases where a virus was the only etiology, and developed predictive models against 2317 cases where the etiology was known but non-viral (bacterial, protozoal, or mixed). Variables predictive of a viral etiology included lower age, a dry and cold season, increased height-for-age z-score (HAZ), lack of bloody diarrhea, and presence of vomiting. Cross-validation suggests an AUC of 0.825 can be achieved with a parsimonious model of 5 variables, achieving a specificity of 0.85, a sensitivity of 0.59, a NPV of 0.82 and a PPV of 0.64.ConclusionPredictors of the etiology of pediatric diarrhea can be used by providers in low-resource settings to inform clinical decision-making. The use of non-laboratory methods to diagnose viral causes of diarrhea could be a step towards reducing inappropriate antibiotic prescription worldwide.

Redefining enteroaggregative Escherichia coli (EAEC): Genomic characterization of epidemiological EAEC strains.

Although enteroaggregative E. coli (EAEC) has been implicated as a common cause of diarrhea in multiple settings, neither its essential genomic nature nor its role as an enteric pathogen are fully understood. The current definition of this pathotype requires demonstration of cellular adherence; a working molecular definition encompasses E. coli which do not harbor the heat-stable or heat-labile toxins of enterotoxigenic E. coli (ETEC) and harbor the genes aaiC, aggR, and/or aatA. In an effort to improve the definition of this pathotype, we report the most definitive characterization of the pan-genome of EAEC to date, applying comparative genomics and functional characterization on a collection of 97 EAEC strains isolated in the course of a multicenter case-control diarrhea study (Global Enteric Multi-Center Study, GEMS). Genomic analysis revealed that the EAEC strains mapped to all phylogenomic groups of E. coli. Circa 70% of strains harbored one of the five described AAF variants; there were no additional AAF variants identified, and strains that lacked an identifiable AAF generally did not have an otherwise complete AggR regulon. An exception was strains that harbored an ETEC colonization factor (CF) CS22, like AAF a member of the chaperone-usher family of adhesins, but not phylogenetically related to the AAF family. Of all genes scored, sepA yielded the strongest association with diarrhea (P = 0.002) followed by the increased serum survival gene, iss (p = 0.026), and the outer membrane protease gene ompT (p = 0.046). Notably, the EAEC genomes harbored several genes characteristically associated with other E. coli pathotypes. Our data suggest that a molecular definition of EAEC could comprise E. coli strains harboring AggR and a complete AAF(I-V) or CS22 gene cluster. Further, it is possible that strains meeting this definition could be both enteric bacteria and urinary/systemic pathogens.

Incidence and etiology of clinically-attended, antibiotic-treated diarrhea among children under five years of age in low- and middle-income countries: Evidence from the Global Enteric Multicenter Study.

Diarrhea is a leading cause of antibiotic consumption among children in low- and middle-income countries. While vaccines may prevent diarrhea infections for which children often receive antibiotics, the contribution of individual enteropathogens to antibiotic use is minimally understood. We used data from the Global Enteric Multicenter Study (GEMS) to estimate pathogen-specific incidence of antibiotic-treated diarrhea among children under five years old residing in six countries of sub-Saharan Africa and South Asia before rotavirus vaccine implementation. GEMS was an age-stratified, individually-matched case-control study. Stool specimens were obtained from children presenting to sentinel health clinics with newly-onset, acute diarrhea (including moderate-to-severe and less-severe diarrhea) as well as matched community controls without diarrhea. We used data from conventional and quantitative molecular diagnostic assays applied to stool specimens to estimate the proportion of antibiotic-treated diarrhea cases attributable to each pathogen. Antibiotics were administered or prescribed to 9,606 of 12,109 moderate-to-severe cases and 1,844 of 3,174 less-severe cases. Across all sites, incidence rates of clinically-attended, antibiotic-treated diarrhea were 12.2 (95% confidence interval: 9.0–17.8), 10.2 (7.4–13.9) and 1.9 (1.3–3.0) episodes per 100 child-years at risk at ages 6 weeks to 11 months, 12–23 months, and 24–59 months, respectively. Based on the recommendation for antibiotic treatment to be reserved for cases with dysentery, we estimated a ratio of 12.6 (8.6–20.8) inappropriately-treated diarrhea cases for each appropriately-treated case. Rotavirus, adenovirus serotypes 40/41, Shigella, sapovirus, Shiga toxin-producing Escherichia coli, and Cryptosporidium were the leading antibiotic-treated diarrhea etiologies. Rotavirus caused 29.2% (24.5–35.2%) of antibiotic-treated cases, including the largest share in both the first and second years of life. Shigella caused 14.9% (11.4–18.9%) of antibiotic-treated cases, and was the leading etiology at ages 24–59 months. Our findings should inform the prioritization of vaccines with the greatest potential to reduce antibiotic exposure among children.

Examination of 189 Campylobacter Species Isolates from the Global Enteric Multicenter Study.

We have examined the draft genomes of 189 Campylobacter species isolates from the Global Enteric Multicenter Study, in which Campylobacter species were identified as significant pathogens.

Pathogen-specific risk of seizure in children with moderate-to-severe diarrhoea: Case control study with follow-up.

To determine the pathogen-specific risk of seizure in under-five children hospitalised with moderate-to-severe diarrhoea (MSD) in rural settings. This was a prospective case-control study with follow-up, conducted in a sentinel facility of Global Enteric Multicenter Study in Mirzapur, a rural community of Bangladesh between 2007 and 2010. Children aged 0-59months who presented with MSD and seizure constituted the cases whereas those who did not have seizure comprised the controls. MSD was defined if the episodes were associated with dehydration or dysentery or required hospitalisation with diarrhoea or dysentery. All enrolled children were followed up at home within 50-90days of enrolment. A total of 64 cases and 128 randomly selected controls formed the analysable dataset. The result of logistic regression analysis after adjusting for potential confounders revealed that shigellosis (Shigella species, OR=5.34, 95% CI=2.37-12.04) particularly S. flexneri (OR=3.34, 95% CI=1.48-7.57), S. flexneri 6 (OR=23.24, 95% CI=2.79-193.85), S. sonnei (OR=6.90, 95% CI=2.34-19.85); norovirus (OR=6.77, 95% CI=1.69-27.11), fever (OR=16.75, 95% CI=1.81-154.70) and loss of consciousness (OR=35.25, 95% CI=1.71-726.20) were the independent risk factors for seizure in MSD children. At enrolment, cases had lower WHZ (P=0.006) compared to their peers, follow-up anthropometrics showed significant improvement in WHZ (P<0.001) and WAZ (P<0.05), whereas deterioration in HAZ (P<0.001) in both cases and controls. Childhood MSD episodes particularly due to Shigella and norovirus are often associated with seizure. Prompt identification and appropriate management of children with shigellosis may reduce occurrence and adverse consequences of seizure linked with MSD.