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Given the challenges in accurately identifying unexposed controls in case-control studies of diarrhoea, we examined diarrhoea incidence, subclinical enteric infections and growth stunting within a reference population in the Global Enteric Multicenter Study, Kenya site. Within 'control' children (0-59 months old without diarrhoea in the 7 days before enrolment, n = 2384), we examined surveys at enrolment and 60-day follow-up, stool at enrolment and a 14-day post-enrolment memory aid for diarrhoea incidence. At enrolment, 19% of controls had ⩾1 enteric pathogen associated with moderate-to-severe diarrhoea ('MSD pathogens') in stool; following enrolment, many reported diarrhoea (27% in 7 days, 39% in 14 days). Controls with and without reported diarrhoea had similar carriage of MSD pathogens at enrolment; however, controls reporting diarrhoea were more likely to report visiting a health facility for diarrhoea (27% vs. 7%) or fever (23% vs. 16%) at follow-up than controls without diarrhoea. Odds of stunting differed by both MSD and 'any' (including non-MSD pathogens) enteric pathogen carriage, but not diarrhoea, suggesting control classification may warrant modification when assessing long-term outcomes. High diarrhoea incidence following enrolment and prevalent carriage of enteric pathogens have implications for sequelae associated with subclinical enteric infections and for design and interpretation of case-control studies examining diarrhoea. [ABSTRACT FROM AUTHOR]

Background: Quantitative molecular assays are increasingly used for detection of enteric viruses., Methods: We compared the clinical severity using the modified Vesikari score (mVS) of enteric viruses detected by conventional assays (enzyme immunoassays [EIAs] for rotavirus and adenovirus 40/41 and conventional polymerase chain reaction for astrovirus, sapovirus, and norovirus) and a quantitative molecular assay (TaqMan Array Card [TAC]) among children aged 0-59 months in the Global Enteric Multicenter Study. For rotavirus and adenovirus 40/41, we compared severity between EIA-positive and TAC-positive cases assigned etiologies using different cycle threshold (Ct) cutoffs., Results: Using conventional assays, the median mVS (interquartile range) was 10 (8-11) for rotavirus, 9 (7-11) for adenovirus 40/41, 8 (6-10) for astrovirus, sapovirus, and norovirus GII, and 7 (6-9) for norovirus GI. Compared with rotavirus EIA-positive cases, the median mVS was 2 and 3 points lower for EIA-negative/TAC-positive cases with Ct <32.6 or Ct ≥32.6 and <35, respectively (P < .001). Adenovirus 40/41 EIA-positive and EIA-negative/TAC-positive cases were similar, regardless of Ct cutoff., Conclusions: Quantitative molecular assays compared with conventional assays, such as EIA, may influence the severity of identified cases, especially for rotavirus. Cutoffs to assign etiology for quantitative assays should be considered in the design and interpretation of enteric virus studies., Competing Interests: Potential conflicts of interest. D. S. reports employment and shares with GSK Vaccines, outside the submitted work. A. K. M. Z. was involved in this work as the site principial investigator of the GEMS study at the Aga Khan University during 2008–2012; she is currently the President of Gender Equality at the Bill & Melinda Gates Foundation and has not influenced this analysis from her position there. K. L. K. received funding from the Bill & Melinda Gates Foundation for part of the submitted work and outside the submitted work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

Peptidylarginine deiminase type4 (PAD4) is a pivotal pro-inflammatory protein within the human immune system, intricately involved in both inflammatory processes and immune responses. Its role extends to the generation of diverse immune cell types, including T cells, B cells, natural killer cells, and dendritic cells. PAD4 has recently garnered attention due to its association with a spectrum of inflammatory and autoimmune disorders, notably rheumatoid arthritis (RA). Mutations in the PAD4 gene, leading to the conversion of arginine to citrulline, have emerged as significant factors in the pathogenesis of RA and related conditions. As a calcium-dependent enzyme, PAD4 is central to the citrullination process, a crucial post-translational modification implicated in disease pathophysiology. Its critical role in autoimmune disorders and inflammation makes PAD4 a prime candidate for therapeutic intervention in RA. Inhibiting PAD4 presents a promising avenue for mitigating inflammatory responses and curtailing joint degradation and impairment. To explore its therapeutic potential, a structure-based virtual screening (SBVS) approach was employed, harnessing an array of marine natural products (MNPs) sourced from databases such as CMNPD, MNPD, and Seaweed. Notably, MNPD10752, CMNPD12680, and CMNPD2751 emerged as potential hit molecules, exhibiting adherence to essential pharmacokinetic properties and favorable toxicity profiles. Quantum mechanics studies using density functional theory (DFT) calculations revealed the inhibitory potential of these identified natural products. Further structural elucidation through molecular dynamics simulations (MDS) and principal component-based free energy landscape (FEL) analysis shed light on the stability of MNP-bound PAD4 complexes. In conclusion, this computational study serves as a stepping stone for further experimental evaluation, aiming to explore the potential of MNPs in addressing PAD4-related human pathologies., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The white spot syndrome virus (WSSV), a rapidly replicating and highly lethal pathogen that targets Penaeid shrimp, has emerged as one of the most widespread viruses globally due to its high virulence. With effective chemotherapeutics still unavailable, the pursuit of novel and viable strategies against WSSV remains a crucial focus in the field of shrimp farming. The envelope proteins of WSSV are essential for virus entry, serving as excellent targets for the development of antiviral therapeutics. Novel strategies in the design of inhibitory peptides, especially those targeting envelope protein (VP28) located on the surface of the virus particle, play a critical role as a significant virulence factor during the early stages of inherent WSSV infection in shrimp. In this direction, the current computational study focused on identifying self-inhibitory peptides from the hydrophobic membrane regions of the VP28 protein, employing peptide docking and molecular dynamics simulation (MDS) approaches. Such inhibitory peptides could be useful building blocks for the rational engineering of inhibitory therapeutics since they imitate the mechanism of binding to homologous partners used by their origin domain to interact with other molecules. The N-terminal sequence of VP28 has been reported as the potential site for membrane interactions during the virus entry. Moreover, drug delivery systems mediated by chitosan and gold nanoparticles are being developed to enhance the therapeutic efficacy of anti-viral peptides. These systems can increase the solubility, stability, and selectivity of peptides, possessing better qualities than conventional delivery methods. This computational study on self-inhibitory peptides could be a valuable resource for further in vitro and in vivo studies on anti-viral therapeutics in the aquaculture industry., Competing Interests: Declaration of competing interest All authors declare that there are no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Biosurfactant based biostimulants plays a vital role in agriculture filed by enhancing the soil quality, promote plant growth, and eliminate plant pathogens, and increasing nutrient uptake. This manuscript describes the synthesis of trimesic based lithocholic ester functionalized amphiphiles (TMLCEA) with oppositely charged head groups using thiol-yne click chemistry, which is an effective and simple approach. The trimesic based lithocholic ester functionalized zwitterionic penicillamine (TMLCEPA), cationic cysteamine·HCl (TMLCECy), and anionic thiomalic acid (TMLCETM) exhibited hierarchically self-assembled microstructures from below to above the CMC. In below the CMC, TMLCEPA, TMLCECy, and TMLCETM showed a bundle of petals, flower-like morphology, and grass seed-like patterns respectively. The morphology of self-assembly was studied by FE-SEM, DLS, OPM, contact angle, and zeta potential measurements. Among these amphiphiles, TMLCECy exhibited potential antimicrobial activity at above the CMC. The biostimulant effect of different concentration of TMLCEA treated with maize and green gram seeds were evaluated under in vitro condition, wherein TMLCECy showed improved seed germination and seedling parameters at 750 µL/mL as compared to TMLCEPA, TMLCETM and untreated amphiphiles as control. Molecular docking and molecular dynamic simulations show that TMLCEPA and TMLCETM showed higher binding affinity for dengue methyltransferase protein. The result of the present study opens up new avenues for bile acid-based amphiphiles as bio-based and cost-effective biostimulants for sustainable agriculture., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The two most serious global health challenges confronting human society today are autoimmune disorders (AIDs) and neurological diseases (NDs), both of which shorten people's lives and worsen the situation. Despite their extensive impact, statistics show that AIDs is associated with a higher risk of ND. Circular RNAs (circRNAs) are critical in several illnesses and disorders, especially AID and ND. Therefore, the present study focused on understanding the underlying causes of the pathophysiology of diseases such as AID and ND through in silico -based research. In order to determine how circRNAs are related to various disease pathways, this study examined the gene expression data sets for Rheumatoid arthritis (RA), Alzheimer's disease (AD), and atrial fibrillation (AF). Our study identified and analyzed two circRNAs, their respective host genes (DHTKD1 and RAN) and their related miRNAs, which could serve as potential markers for treating disorders like myotonic dystrophy type 1, spinocerebellar ataxia and fragile X syndrome. Further, the circRNA-derived peptide was identified and analysed with the molecular dynamics simulation (MDS) followed by a principal component (PC) based free energy landscape (FEL) profile. The computational results obtained here provide a basis for the development of therapeutics against AD, RA and AF. Moreover, further functional studies are needed to validate their role in disease aetiology and to provide a detailed understanding of their association with AID and ND.Communicated by Ramaswamy H. Sarma.

Amyotrophic lateral sclerosis is a neurodegenerative disease that damages motor neurons and causes gradual muscular weakening and paralysis. Although studies have linked a number of genetic and environmental factors to ALS, the specific causes and mechanisms of the disease are still unclear. The pivotal role of circular RNA in the pathogenesis of ALS is a newly emerging area of research. The term "circular RNA" describes a particular class of RNA molecule that, in contrast to most RNA molecules, has a closed-loop structure. According to recent research, circular RNA might be essential for the development and progression of ALS. It has been discovered that these circular RNAs support important cellular functions related to ALS, including protein turnover, mitochondrial function, RNA processing, and cellular transport. Gaining knowledge about the precise roles and processes of circular RNA in the development of ALS could assist in understanding the pathophysiology of the disease and possibly pave the way for the development of targeted therapies. However, the understanding of circular RNA in ALS is still limited, and more research is needed to fully elucidate its role. In order to gain a comprehensive understanding of the role of circRNAs in ALS, it is imperative to delve into the various mechanisms through which circRNAs may contribute to the development and progression of the disease. Examining the current status of circRNA research in ALS and offering insights into their potential as therapeutic targets and diagnostic markers are the primary objectives of this review., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Due to its emerging resistance to first-line anti-TB medications, tuberculosis (TB) is one of the most contagious illness in the world. According to reports, the effectiveness of treating TB is severely impacted by drug resistance, notably resistance caused by mutations in the pncA gene-encoded pyrazinamidase (PZase) to the front-line drug pyrazinamide (PZA). The present study focused on investigating the resistance mechanism caused by the mutations D12N, T47A, and H137R to better understand the structural and molecular events responsible for the resistance acquired by the pncA gene of Mycobacterium tuberculosis (MTB) at the structural level. Bioinformatics analysis predicted that all three mutations were deleterious and located near the active centre of the pncA, affecting its functional activity. Furthermore, molecular dynamics simulation (MDS) results established that mutations significantly reduced the structural stability and caused the rearrangement of FE 2+ in the active centre of pncA. Moreover, essential dynamics analysis, including principal component analysis (PCA) and free energy landscape (FEL), concluded variations in the protein motion and decreased conformational space in the mutants. Additionally, the mutations potentially impacted the network topologies and altered the residual communications in the network. The complex simulation study results established the significant movement of the flap region from the active centre of mutant complexes, further supporting the flap region's significance in developing resistance to the PZA drug. This study advances our knowledge of the primary cause of the mechanism of PZA resistance and the structural dynamics of pncA mutants, which will help us to design new and potent chemical scaffolds to treat drug-resistant TB (DR-TB)., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) is a lethal multisystem disease that attacks the lungs' first line of defense. A substantial threat to public health and a primary cause of death is pulmonary TB. This study aimed to identify and investigate the probable differentially expressed genes (DEGs) primarily involved in Pulmonary TB. Accordingly, three independent gene expression data sets, numbered GSE139825, GSE139871, and GSE54992, were utilized for this purpose. The identified DEGs were used for bioinformatics-based analysis, including physical gene interaction, Gene Ontology (GO), network analysis and pathway studies using the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG). The computational analysis predicted that TNFAIP6 is the significant DEG in the gene expression profiling of TB datasets. According to gene ontology analysis, TNFAIP6 is also essential in injury and inflammation. Further, TNFA1P6 is strongly linked to arsenic poisoning, evident from the results of NetworkAnalyst, a comprehensive and interactive platform for gene expression profiling via network visual analytics. As a result, the TNFAIP6 gene was ultimately chosen as a candidate DEG and subsequently employed for in silico structural characterization studies. The tertiary structure of TNFAIP6 was modelled using the ROBETTA server, followed by validation with SAVES and ProSA webserver. Additionally, structural dynamic studies, including molecular dynamics simulation (MDS) and essential dynamics analysis, including principal component (PC) based free energy landscape (FEL) analysis, was used for checking the stability of TNFAIP6 models. The dynamics result established the structural rigidity of modelled TNFAIP6 through RMSD, RMSF and RoG results. The FEL analysis revealed the restricted conformational flexibility of TNFAIP6 by displaying a single minimum energy basin in the contour plot. The comprehensive computational analysis established that TNFAIP6 could serve as a viable biomarker to assess the severity of pulmonary TB., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: DNP Scholarly projects require review for scientific merit and human subject protection. Rapid growth of DNP programs and projects has increased Institutional Review Board (IRB) burden and increased the length of project approval time when most DNP scholarly projects are quality improvement (QI) projects and not deemed Human Subjects Research (HSR)., Purpose: Develop a process and describe the rationale for creating and implementing a Project Ethical Review Committee (PERC) in the School of Nursing and to evaluate the experience of the first cohort of submissions., Process: Committee was formed using expert consensus approach, in collaboration with IRB. Standards of Procedures and training materials created., Outcome Measure: 100 projects submitted to committee; 95 deemed QI (95%) and 5 projects considered HSR (5%). First 94 projects were reviewed, and approval letters sent within 8 weeks., Discussion: This paper discusses how PERC ensures rigorous and ethical review process for standardization, timeliness, and efficiency., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The 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., Methods: The 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., Results: Among 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)., Conclusions: Linear 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., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: We present findings from the Pneumonia Etiology Research for Child Health (PERCH) site in Bamako, Mali., Methods: Cases were patients 28 days to 59 months of age, admitted to hospital with severe or very severe pneumonia (2005 World Health Organization definition). Community controls were frequency matched by age. Both provided nasopharyngeal and oropharyngeal swabs for multiplex polymerase chain reaction and Streptococcus pneumoniae culture. Cases underwent blood culture and induced sputum culture for Mycobacterium tuberculosis. A subset had pleural fluid and lung aspirates collected for culture and polymerase chain reaction. Primary analyses included participants with negative or unknown HIV status (HIV-) and cases with abnormal chest radiographs (CXR+). Cases and controls were compared using logistic regression adjusting for age. Etiologic fractions were calculated by a Bayesian nested partially latent class analysis, the PERCH integrated analysis., Results: Between January 1, 2012, and January 14, 2014, we enrolled 241 CXR+/HIV- cases and 725 HIV- controls. Compared with controls, cases were more likely to have moderate-to-severe wasting (43.1% vs. 14.1%, P < 0.001) and stunting (26.6% vs. 9.4%, P < 0.001). Predominant etiologies were respiratory syncytial virus [24.0%; 95% credible interval (CrI): 18.3%-31.1%], S. pneumoniae (15.2%; 95% CrI: 9.5-21.6), human metapneumovirus (11.8%; 95% CrI: 8.3%-16.2%) and parainfluenza virus type 3 (9.0%; 95% CrI: 5.8%-13.3%). Case fatality was 13.3%, with Staphylococcus aureus, Pneumocystis jirovecii and Haemophilus influenzae type b predominating (40% of fatal cases)., Conclusions: PERCH uncovered high case fatality among children with severe pneumonia in Mali, highlighting a role for new interventions (eg, respiratory syncytial virus vaccines) and a need to improve vaccine coverage and strengthen healthcare delivery., Competing Interests: The authors have no conflicts of interest to disclose. M.D.K. has received funding for consultancies from Merck, Pfizer and Novartis and grant funding from Merck. M.M.H. has received grant funding from Pfizer. L.L.H. has received grant funding from Pfizer and GlaxoSmithKline. C.P. has received grant funding from Merck. K.L.O. has received grant funding from GlaxoSmithKline and Pfizer and participated on technical advisory boards for Merck, Sanofi-Pasteur, PATH, Affinivax and ClearPath. K.L.K. has received grant funding from Merck Sharp & Dohme. The other authors have no funding or conflicts of interest to disclose., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

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., Methods: We 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., Results: Compared 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., Conclusions: A 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., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Rotavirus 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., Methods: A 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., Results: Overall 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., Conclusion: The implementation of vaccination against rotavirus may likely result in a significant reduction of rotavirus-associated diarrhea, suggesting the need for monitoring of vaccine impact., Competing Interests: The authors have no conflict of interest

Background: Enterotoxigenic Escherichia coli (ETEC) encoding heat-stable enterotoxin (ST) alone or with heat-labile enterotoxin (LT) cause moderate-to-severe diarrhea (MSD) in developing country children. The Global Enteric Multicenter Study (GEMS) identified ETEC encoding ST among the top four enteropathogens. Since the GEMS objective was to provide evidence to guide development and implementation of enteric vaccines and other interventions to diminish diarrheal disease morbidity and mortality, we examined colonization factor (CF) prevalence among ETEC isolates from children age <5 years with MSD and from matched controls in four African and three Asian sites. We also assessed strength of association of specific CFs with MSD., Methodology/principal Findings: MSD cases enrolled at healthcare facilities over three years and matched controls were tested in a standardized manner for many enteropathogens. To identify ETEC, three E. coli colonies per child were tested by polymerase chain reaction (PCR) to detect genes encoding LT, ST; confirmed ETEC were examined by PCR for major CFs (Colonization Factor Antigen I [CFA/I] or Coli Surface [CS] antigens CS1-CS6) and minor CFs (CS7, CS12, CS13, CS14, CS17, CS18, CS19, CS20, CS21, CS30). ETEC from 806 cases had a single toxin/CF profile in three tested strains per child. Major CFs, components of multiple ETEC vaccine candidates, were detected in 66.0% of LT/ST and ST-only cases and were associated with MSD versus matched controls by conditional logistic regression (p≤0.006); major CFs detected in only 25.0% of LT-only cases weren't associated with MSD. ETEC encoding exclusively CS14, identified among 19.9% of 291 ST-only and 1.5% of 259 LT/ST strains, were associated with MSD (p = 0.0011). No other minor CF exhibited prevalence ≥5% and significant association with MSD., Conclusions/significance: Major CF-based efficacious ETEC vaccines could potentially prevent up to 66% of pediatric MSD cases due to ST-encoding ETEC in developing countries; adding CS14 extends coverage to ~77%., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests. We note that the following authors are involved in the development of vaccines to prevent diarrhea caused by enterotoxigenic Escherichia coli: James P. Nataro: Has been named in patents for technology that may have relevance for ETEC vaccine technology; JPN is also a co-investigator on grants from non-profit agencies that support ETEC vaccine development. Halvor Sommerfelt: Has been named in patents for technology that may have relevance for ETEC vaccine technology including: INT Application Number PCT/IB2014/000267; INT Publication Number WO2014128555 A2 “ETEC Vaccine”. European Patent Application No. 14711297.3 United States Patent Application No. 14/769,342. China Patent Application No. 201480022329.6. Puntervoll P, Sommerfelt H, Clements J, Nataro JP, Zhang W, Taxt A. HS is also a co-investigator on grants from non-profit agencies that support ETEC vaccine development Ann-Mari Svennerholm: Has shares in the University of Göteborg spin-out biotech company Gotovax AB which is entitled to royalty from Scandinavian Biopharma on sales in travelers of the ETEC vaccine Etvax if it becomes a commercial product. A-MS is a co-inventor on ETEC vaccine patent application owned by Scandinavian Biopharma and has a research grant from the Swedish Foundation for Strategic Research for infection biology research. Myron M. Levine: Is a co-inventor of patents for ETEC vaccine technology including US patent #6,902,736 B2. “Isolation and characterization of the CSA operon (ETEC-CS4 pili) and methods of using same.” He is a member of the Scientific Advisory Board of the PaxVax Corporation. MML is also the recipient for grants relevant to ETEC vaccine development including the Enteric Center of Excellence for Translational Research (Enteric-CETR) “Immunoprophylactic Strategies to Control Emerging Enteric Infections” (NIAID U19AI109776; MML, PI)

Background: Cryptosporidium is a leading cause of moderate-to-severe diarrhea (MSD) in young children in Africa. We examined factors associated with Cryptosporidium infection in MSD cases enrolled at the rural western Kenya Global Enteric Multicenter Study (GEMS) site from 2008-2012., Methodology/principal Findings: At health facility enrollment, stool samples were tested for enteric pathogens and data on clinical, environmental, and behavioral characteristics collected. Each child's health status was recorded at 60-day follow-up. Data were analyzed using logistic regression. Of the 1,778 children with MSD enrolled as cases in the GEMS-Kenya case-control study, 11% had Cryptosporidium detected in stool by enzyme immunoassay; in a genotyped subset, 81% were C. hominis. Among MSD cases, being an infant, having mucus in stool, and having prolonged/persistent duration diarrhea were associated with being Cryptosporidium-positive. Both boiling drinking water and using rainwater as the main drinking water source were protective factors for being Cryptosporidium-positive. At follow-up, Cryptosporidium-positive cases had increased odds of being stunted (adjusted odds ratio [aOR] = 1.65, 95% CI: 1.06-2.57), underweight (aOR = 2.08, 95% CI: 1.34-3.22), or wasted (aOR = 2.04, 95% CI: 1.21-3.43), and had significantly larger negative changes in height- and weight-for-age z-scores from enrollment., Conclusions/significance: Cryptosporidium contributes significantly to diarrheal illness in young children in western Kenya. Advances in point of care detection, prevention/control approaches, effective water treatment technologies, and clinical management options for children with cryptosporidiosis are needed., Competing Interests: The authors have declared that no competing interests exist.

Diarrheal disease is a leading cause of death among young children worldwide. As rates of acute diarrhea (AD; 1-6 days duration) have decreased, persistent diarrhea (PD; > 14 days duration) accounts for a greater proportion of the diarrheal disease burden. We describe factors associated with the duration of moderate-to-severe diarrhea in Kenyan children < 5 years old enrolled in the Global Enteric Multicenter Study. We found 587 (58%) children experienced AD, 360 (35%) had prolonged acute diarrhea (ProAD; 7-13 days duration), and 73 (7%) had PD. We constructed a Cox proportional hazards model to identify factors associated with diarrheal duration. Risk factors independently associated with longer diarrheal duration included infection with Cryptosporidium (hazard ratio [HR]: 0.868, P = 0.035), using an unimproved drinking water source (HR: 0.87, P = 0.035), and being stunted at enrollment (HR: 0.026, P < 0.0001). Diarrheal illness of extended duration appears to be multifactorial; given its association with adverse health and development outcomes, effective strategies should be implemented to reduce the duration and severity of diarrheal illness. Effective treatments for Cryptosporidium should be identified, interventions to improve drinking water are imperative, and nutrition should be improved through exclusive breastfeeding in infants ≤ 6 months and appropriate continued feeding practices for ill children.

Background.: It is standard practice for laboratories to assess the cellular quality of expectorated sputum specimens to check that they originated from the lower respiratory tract. The presence of low numbers of squamous epithelial cells (SECs) and high numbers of polymorphonuclear (PMN) cells are regarded as indicative of a lower respiratory tract specimen. However, these quality ratings have never been evaluated for induced sputum specimens from children with suspected pneumonia., Methods.: We evaluated induced sputum Gram stain smears and cultures from hospitalized children aged 1-59 months enrolled in a large study of community-acquired pneumonia. We hypothesized that a specimen representative of the lower respiratory tract will contain smaller quantities of oropharyngeal flora and be more likely to have a predominance of potential pathogens compared to a specimen containing mainly saliva. The prevalence of potential pathogens cultured from induced sputum specimens and quantity of oropharyngeal flora were compared for different quantities of SECs and PMNs., Results.: Of 3772 induced sputum specimens, 2608 (69%) had <10 SECs per low-power field (LPF) and 2350 (62%) had >25 PMNs per LPF, measures traditionally associated with specimens from the lower respiratory tract in adults. Using isolation of low quantities of oropharyngeal flora and higher prevalence of potential pathogens as markers of higher quality, <10 SECs per LPF (but not >25 PMNs per LPF) was the microscopic variable most associated with high quality of induced sputum., Conclusions.: Quantity of SECs may be a useful quality measure of induced sputum from young children with pneumonia., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

The Pneumonia Etiology Research for Child Health study was conducted across 7 diverse research sites and relied on standardized clinical and laboratory methods for the accurate and meaningful interpretation of pneumonia etiology data. Blood, respiratory specimens, and urine were collected from children aged 1-59 months hospitalized with severe or very severe pneumonia and community controls of the same age without severe pneumonia and were tested with an extensive array of laboratory diagnostic tests. A standardized testing algorithm and standard operating procedures were applied across all study sites. Site laboratories received uniform training, equipment, and reagents for core testing methods. Standardization was further assured by routine teleconferences, in-person meetings, site monitoring visits, and internal and external quality assurance testing. Targeted confirmatory testing and testing by specialized assays were done at a central reference laboratory., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

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 o 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To evaluate factors associated with rotavirus diarrhea and to describe severity of illness among children <5 years old with non-dysenteric, moderate-to-severe diarrhea (MSD) in rural western Kenya., Methods: We analyzed data from children <5 years old with non-dysenteric MSD enrolled as cases in the Global Enteric Multicenter Study (GEMS) in Kenya. A non-dysenteric MSD case was defined as a child with ≥3 loose stools in 24 hrs. and one or more of the following: sunken eyes, skin tenting, intravenous rehydration, or hospitalization, who sought care at a sentinel health center within 7 days of illness onset. Rotavirus antigens in stool samples were detected by ELISA. Demographic and clinical information was collected at enrollment and during a single follow-up home visit at approximately 60 days. We analyzed diarrhea severity using a GEMS 17 point numerical scoring system adapted from the Vesikari score. We used logistic regression to evaluate factors associated with rotavirus infection., Results: From January 31, 2008 to September 30, 2012, among 1,637 (92%) non-dysenteric MSD cases, rotavirus was detected in stools of 245 (15.0%). Rotavirus-positive compared with negative cases were: younger (median age, 8 vs. 13 months; p<0.0001), had more severe illness (median severity score, 9 vs 8; p<0.0001) and had to be hospitalized more frequently (37/245 [15.1%] vs. 134/1,392 [9.6%]), p <0.013). Independent factors associated with rotavirus infection included age 0-11 months old (aOR = 5.29, 95% CI 3.14-8.89) and presenting with vomiting ≥3 times/24hrs (aOR = 2.58, 95% CI [1.91-3.48]). Rotavirus was detected more commonly in warm and dry months than in the cool and rainy months (142/691 [20%] vs 70/673 [10%]) p<0.0001)., Conclusions: Diarrhea caused by rotavirus is associated with severe symptoms leading to hospitalization. Consistent with other settings, infants had the greatest burden of disease.

Background: The importance of Cryptosporidium as a pediatric enteropathogen in developing countries is recognized., Methods: Data from the Global Enteric Multicenter Study (GEMS), a 3-year, 7-site, case-control study of moderate-to-severe diarrhea (MSD) and GEMS-1A (1-year study of MSD and less-severe diarrhea [LSD]) were analyzed. Stools from 12,110 MSD and 3,174 LSD cases among children aged <60 months and from 21,527 randomly-selected controls matched by age, sex and community were immunoassay-tested for Cryptosporidium. Species of a subset of Cryptosporidium-positive specimens were identified by PCR; GP60 sequencing identified anthroponotic C. parvum. Combined annual Cryptosporidium-attributable diarrhea incidences among children aged <24 months for African and Asian GEMS sites were extrapolated to sub-Saharan Africa and South Asian regions to estimate region-wide MSD and LSD burdens. Attributable and excess mortality due to Cryptosporidium diarrhea were estimated., Findings: Cryptosporidium was significantly associated with MSD and LSD below age 24 months. Among Cryptosporidium-positive MSD cases, C. hominis was detected in 77.8% (95% CI, 73.0%-81.9%) and C. parvum in 9.9% (95% CI, 7.1%-13.6%); 92% of C. parvum tested were anthroponotic genotypes. Annual Cryptosporidium-attributable MSD incidence was 3.48 (95% CI, 2.27-4.67) and 3.18 (95% CI, 1.85-4.52) per 100 child-years in African and Asian infants, respectively, and 1.41 (95% CI, 0.73-2.08) and 1.36 (95% CI, 0.66-2.05) per 100 child-years in toddlers. Corresponding Cryptosporidium-attributable LSD incidences per 100 child-years were 2.52 (95% CI, 0.33-5.01) and 4.88 (95% CI, 0.82-8.92) in infants and 4.04 (95% CI, 0.56-7.51) and 4.71 (95% CI, 0.24-9.18) in toddlers. We estimate 2.9 and 4.7 million Cryptosporidium-attributable cases annually in children aged <24 months in the sub-Saharan Africa and India/Pakistan/Bangladesh/Nepal/Afghanistan regions, respectively, and ~202,000 Cryptosporidium-attributable deaths (regions combined). ~59,000 excess deaths occurred among Cryptosporidium-attributable diarrhea cases over expected if cases had been Cryptosporidium-negative., Conclusions: The enormous African/Asian Cryptosporidium disease burden warrants investments to develop vaccines, diagnostics and therapies.

Enteropathogenic Escherichia coli (EPEC) are diarrhoeagenic E. coli, and are a significant cause of gastrointestinal illness among young children in developing countries. Typical EPEC are identified by the presence of the bundle-forming pilus encoded by a virulence plasmid, which has been linked to an increased severity of illness, while atypical EPEC lack this feature. Comparative genomics of 70 total EPEC from lethal (LI), non-lethal symptomatic (NSI) or asymptomatic (AI) cases of diarrhoeal illness in children enrolled in the Global Enteric Multicenter Study was used to investigate the genomic differences in EPEC isolates obtained from individuals with various clinical outcomes. A comparison of the genomes of isolates from different clinical outcomes identified genes that were significantly more prevalent in EPEC isolates of symptomatic and lethal outcomes than in EPEC isolates of asymptomatic outcomes. These EPEC isolates exhibited previously unappreciated phylogenomic diversity and combinations of virulence factors. These comparative results highlight the diversity of the pathogen, as well as the complexity of the EPEC virulence factor repertoire.

Molecular identification of the invasion plasmid antigen-H (ipaH) gene has been established as a useful detection mechanism for Shigella spp. The Global Enteric Multicenter Study (GEMS) identified the etiology and burden of moderate-to-severe diarrhea (MSD) in sub-Saharan Africa and south Asia using a case-control study and traditional culture techniques. Here, we used quantitative polymerase chain reaction (qPCR) to identify Shigella spp. in 2,611 stool specimens from GEMS and compared these results to those using culture. Demographic and nutritional characteristics were assessed as possible risk factors. The qPCR identified more cases of shigellosis than culture; however, the distribution of demographic characteristics was similar by both methods. In regression models adjusting for Shigella quantity, age, and site, children who were exclusively breast-fed had significantly lower odds of MSD compared with children who were not breast-fed (odds ratio [OR] = 0.47, 95% confidence interval (CI) = 0.28-0.81). The association between Shigella quantity and MSD increased with age, with a peak in children of 24-35 months of age (OR = 8.2, 95% CI = 4.3-15.7) and the relationship between Shigella quantity and disease was greatest in Bangladesh (OR = 13.2, 95% CI = 7.3-23.8). This study found that qPCR identified more cases of Shigella and age, site, and breast-feeding status were significant risk factors for MSD., (© The American Society of Tropical Medicine and Hygiene.)

Background: Typical enteropathogenic Escherichia coli (tEPEC) strains were associated with mortality in the Global Enteric Multicenter Study (GEMS). Genetic differences in tEPEC strains could underlie some of the variability in clinical outcome., Methods: We produced draft genome sequences of all available tEPEC strains from GEMS lethal infections (LIs) and of closely matched EPEC strains from GEMS subjects with non-lethal symptomatic infections (NSIs) and asymptomatic infections (AIs) to identify gene clusters (potential protein encoding sequences sharing ≥90% nucleotide sequence identity) associated with lethality., Results: Among 14,412 gene clusters identified, the presence or absence of 392 was associated with clinical outcome. As expected, more gene clusters were associated with LI versus AI than LI versus NSI. The gene clusters more prevalent in strains from LI than those from NSI and AI included those encoding proteins involved in O-antigen biogenesis, while clusters encoding type 3 secretion effectors EspJ and OspB were among those more prevalent in strains from non-lethal infections. One gene cluster encoding a variant of an NleG ubiquitin ligase was associated with LI versus AI, while two other nleG clusters had the opposite association. Similar associations were found for two nleG gene clusters in an additional, larger sample of NSI and AI GEMS strains., Conclusions: Particular genes are associated with lethal tEPEC infections. Further study of these factors holds potential to unravel the mechanisms underlying severe disease and to prevent adverse outcomes.

Pathogens in the gastrointestinal tract exist within a vast population of microbes. We examined associations between pathogens and composition of gut microbiota as they relate to Shigella spp./enteroinvasive Escherichia coli infection. We analyzed 3,035 stool specimens (1,735 nondiarrheal and 1,300 moderate-to-severe diarrheal) from the Global Enteric Multicenter Study for 9 enteropathogens. Diarrheal specimens had a higher number of enteropathogens (diarrheal mean 1.4, nondiarrheal mean 0.95; p<0.0001). Rotavirus showed a negative association with Shigella spp. in cases of diarrhea (odds ratio 0.31, 95% CI 0.17-0.55) and had a large combined effect on moderate-to-severe diarrhea (odds ratio 29, 95% CI 3.8-220). In 4 Lactobacillus taxa identified by 16S rRNA gene sequencing, the association between pathogen and disease was decreased, which is consistent with the possibility that Lactobacillus spp. are protective against Shigella spp.-induced diarrhea. Bacterial diversity of gut microbiota was associated with diarrhea status, not high levels of the Shigella spp. ipaH gene.

Background: Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease., Results: We use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age., Conclusions: Our findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.

Water, sanitation, and hygiene information was collected during a matched case-control study of moderate and severe diarrhea (MSD) among 4,096 children < 5 years of age in Bamako, Mali. Primary use of piped water (conditional odds ratio [cOR] = 0.45; 0.34-0.62), continuous water access (cOR = 0.30; 0.20-0.43), fetching water daily (cOR = 0.77; 0.63-0.96), and breastfeeding (cOR = 0.65; 0.49-0.88) significantly reduced the likelihood of MSD. Fetching water in > 30 minutes (cOR = 2.56; 1.55-4.23) was associated with MSD. Piped tap water and courier-delivered water contained high (> 2 mg/L) concentrations of free residual chlorine and no detectable Escherichia coli. However, many households stored water overnight, resulting in inadequate free residual chlorine (< 0.2 mg/L) for preventing microbial contamination. Coliforms and E. coli were detected in 48% and 8% of stored household water samples, respectively. Although most of Bamako's population enjoys access to an improved water source, water quality is often compromised during household storage.

Background: Diarrhoeal diseases cause illness and death among children younger than 5 years in low-income countries. We designed the Global Enteric Multicenter Study (GEMS) to identify the aetiology and population-based burden of paediatric diarrhoeal disease in sub-Saharan Africa and south Asia., Methods: The GEMS is a 3-year, prospective, age-stratified, matched case-control study of moderate-to-severe diarrhoea in children aged 0-59 months residing in censused populations at four sites in Africa and three in Asia. We recruited children with moderate-to-severe diarrhoea seeking care at health centres along with one to three randomly selected matched community control children without diarrhoea. From patients with moderate-to-severe diarrhoea and controls, we obtained clinical and epidemiological data, anthropometric measurements, and a faecal sample to identify enteropathogens at enrolment; one follow-up home visit was made about 60 days later to ascertain vital status, clinical outcome, and interval growth., Findings: We enrolled 9439 children with moderate-to-severe diarrhoea and 13,129 control children without diarrhoea. By analysing adjusted population attributable fractions, most attributable cases of moderate-to-severe diarrhoea were due to four pathogens: rotavirus, Cryptosporidium, enterotoxigenic Escherichia coli producing heat-stable toxin (ST-ETEC; with or without co-expression of heat-labile enterotoxin), and Shigella. Other pathogens were important in selected sites (eg, Aeromonas, Vibrio cholerae O1, Campylobacter jejuni). Odds of dying during follow-up were 8·5-fold higher in patients with moderate-to-severe diarrhoea than in controls (odd ratio 8·5, 95% CI 5·8-12·5, p<0·0001); most deaths (167 [87·9%]) occurred during the first 2 years of life. Pathogens associated with increased risk of case death were ST-ETEC (hazard ratio [HR] 1·9; 0·99-3·5) and typical enteropathogenic E coli (HR 2·6; 1·6-4·1) in infants aged 0-11 months, and Cryptosporidium (HR 2·3; 1·3-4·3) in toddlers aged 12-23 months., Interpretation: Interventions targeting five pathogens (rotavirus, Shigella, ST-ETEC, Cryptosporidium, typical enteropathogenic E coli) can substantially reduce the burden of moderate-to-severe diarrhoea. New methods and accelerated implementation of existing interventions (rotavirus vaccine and zinc) are needed to prevent disease and improve outcomes., Funding: The Bill & Melinda Gates Foundation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥ 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10(4) ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.