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Background: Environmental Enteric Dysfunction (EED) is an acquired disorder of asymptomatic altered gut function, the etiology of which is unknown. EED is postulated to be a major contributor to growth faltering in early childhood in regions where early-life enteropathogenic carriage is prevalent. Few studies have examined the critical organ (the upper small bowel) with enteropathogens in the evolution of small bowel disease., Objectives: The objective of this study was to determine if fecal enteropathogenic detection predicts subsequent EED histology., Methods: Fecal samples were obtained from undernourished children aged <2 y without diarrhea enrolled in 3 cohort studies, who failed nutritional intervention and subsequently underwent endoscopy. Duodenal biopsies from 245 (Bangladesh n = 120, Pakistan n = 57, and Zambia n = 68) children were scored using a semiquantitative histologic grading protocol. Thirteen enteropathogens were sought in common across the 3 centers using TaqMan array cards (TAC) (Bangladesh and Pakistan) and the Luminex platform (Zambia). An additional 18 pathogens and 32 virulence loci were sought by TAC and included in sensitivity analyses restricted to TAC data., Results: Multivariable linear regressions adjusting for study center, age at stool collection, and stool-to-biopsy interval demonstrated the following: 1) an association of norovirus and Shigella detection with subsequent enterocyte injury [β 0.2 (95% CI: 0.1, 0.3); P = 0.002 and β 0.2 (95% CI: 0.0, 0.3); P = 0.008, respectively], 2) association of Campylobacter with intraepithelial lymphocytes [β 0.2 (95% CI: 0.0, 0.4); P = 0.046], and 3) association of Campylobacter and enterotoxigenic Escherichia coli with a summative EED histopathology index score [β 4.2 (95% CI: 0.8, 7.7); P = 0.017 and β 3.9 (95% CI: 0.5, 7.3); P = 0.027, respectively]. All but 2 of these associations (Shigella-enterocyte injury and Campylobacter-index score) were also demonstrated in TAC-only sensitivity analyses, which identified additional associations between other pathogens, pathogen burden, or virulence loci primarily with the same histologic parameters., Conclusions: The detection of some enteropathogens in asymptomatic infections is associated with subsequent EED histopathology. These novel findings offer a basis for future EED etiology and pathogenesis studies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Validated biomarkers could catalyze environmental enteric dysfunction (EED) research., Objectives: Leveraging an EED histology scoring system, this multicountry analysis examined biomarker associations with duodenal histology features among children with EED. We also examined differences in 2-h compared with 1-h urine collections in the lactulose rhamnose (LR) dual sugar test., Methods: Three cohorts of undernourished children unresponsive to nutrition intervention underwent esophagogastroduodenoscopy and duodenal biopsies. Histopathology scores were compared to fecal calprotectin (CAL), myeloperoxidase (MPO), neopterin (NEO), and urinary LR ratio and lactulose percentage recovery. Log-transformed biomarkers were used in linear regressions adjusted for age, center, and sample collection-biopsy time interval in multivariable models., Results: Data on >1 biomarker were available for 120 Bangladeshi (CAL, MPO, NEO, and LR), 63 Pakistani (MPO, NEO, and LR), and 63 Zambian children (CAL). Median age at endoscopy was similar (19 mo) across centers. Median sample collection prior to endoscopy was consistent with each center's study design: 2 wk in Bangladesh (urine and stool) and Zambia (stool), and 6 (urine) and 11 (stool) mo in Pakistan. In multivariable models, intraepithelial lymphocytes were associated with CAL (exponentiated [exp.] coefficient: 1.19; 95% confidence interval [CI]: 1, 1.41), intramucosal Brunner's glands with MPO (exp. coefficient: 1.33; 95% CI: 1.05, 1.69) and NEO (exp. coefficient: 1.37; 95% CI: 1.1, 1.7), and chronic inflammation with NEO (exp. coefficient: 1.61; 95% CI: 1.17, 2.17). Intraepithelial lymphocytes were associated with lactulose % recovery (exp. coefficient: 1.22; 95% CI: 1.05, 1.41). LR recovery was substantially lower in 1-h collections than in 2-h collections., Conclusions: Four commonly used markers of enteric dysfunction were associated with specific histologic features. One-hour urine collection may be insufficient to reflect small bowel permeability in LR testing. While acknowledging the challenges with obtaining relevant tissue, these findings form the basis for further EED biomarker validation research., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Environmental enteric dysfunction (EED) is a subclinical syndrome of altered small intestinal function postulated to be an important contributor to childhood undernutrition. The role of small intestinal bacterial communities in the pathophysiology of EED is poorly defined due to a paucity of studies where there has been a direct collection of small intestinal samples from undernourished children. Sixty-three members of a Pakistani cohort identified as being acutely malnourished between 3 and 6 months of age and whose wasting (weight-for-length Z -score [WLZ]) failed to improve after a 2-month nutritional intervention underwent esophagogastroduodenoscopy (EGD). Paired duodenal luminal aspirates and duodenal mucosal biopsies were obtained from 43 children. Duodenal microbiota composition was characterized by sequencing bacterial 16S rRNA gene amplicons. Levels of bacterial taxa (amplicon sequence variants [ASVs]) were referenced to anthropometric indices, histopathologic severity in biopsies, expression of selected genes in the duodenal mucosa, and fecal levels of an immunoinflammatory biomarker (lipocalin-2). A "core" group of eight bacterial ASVs was present in the duodenal samples of 69% of participants. Streptococcus anginosus was the most prevalent, followed by Streptococcus sp., Gemella haemolysans , Streptococcus australis , Granulicatella elegans , Granulicatella adiacens, and Abiotrophia defectiva . At the time of EGD, none of the core taxa were significantly correlated with WLZ. Statistically significant correlations were documented between the abundances of Granulicatella elegans and Granulicatella adiacens and the expression of duodenal mucosal genes involved in immune responses (dual oxidase maturation factor 2, serum amyloid A, and granzyme H). These results suggest that a potential role for members of the oral microbiota in pathogenesis, notably Streptococcus , Gemella, and Granulicatella species, warrants further investigation.IMPORTANCEUndernutrition among women and children is a pressing global health problem. Environmental enteric dysfunction (EED) is a disease of the small intestine (SI) associated with impaired gut mucosal barrier function and reduced capacity for nutrient absorption. The cause of EED is ill-defined. One emerging hypothesis is that alterations in the SI microbiota contribute to EED. We performed a culture-independent analysis of the SI microbiota of a cohort of Pakistani children with undernutrition who had failed a standard nutritional intervention, underwent upper gastrointestinal tract endoscopy, and had histologic evidence of EED in their duodenal mucosal biopsies. The results revealed a shared group of bacterial taxa in their duodenums whose absolute abundances were correlated with levels of the expression of genes in the duodenal mucosa that are involved in inflammatory responses. A number of these bacterial taxa are more typically found in the oral microbiota, a finding that has potential physiologic and therapeutic implications., Competing Interests: The authors declare no conflict of interest.

BACKGROUNDThe use of high-throughput technologies has enabled rapid advancement in the knowledge of host immune responses to pathogens. Our objective was to compare the repertoire, protection, and maternal factors associated with human milk antibodies to infectious pathogens in different economic and geographic locations.METHODSUsing multipathogen protein microarrays, 878 milk and 94 paired serum samples collected from 695 women in 5 high and low-to-middle income countries (Bangladesh, Finland, Peru, Pakistan, and the United States) were assessed for specific IgA and IgG antibodies to 1,607 proteins from 30 enteric, respiratory, and bloodborne pathogens.RESULTSThe antibody coverage across enteric and respiratory pathogens was highest in Bangladeshi and Pakistani cohorts and lowest in the U.S. and Finland. While some pathogens induced a dominant IgA response (Campylobacter, Klebsiella, Acinetobacter, Cryptosporidium, and pertussis), others elicited both IgA and IgG antibodies in milk and serum, possibly related to the invasiveness of the infection (Shigella, enteropathogenic E. coli "EPEC", Streptococcus pneumoniae, Staphylococcus aureus, and Group B Streptococcus). Besides the differences between economic regions and decreases in concentrations over time, human milk IgA and IgG antibody concentrations were lower in mothers with high BMI and higher parity, respectively. In Bangladeshi infants, a higher specific IgA concentration in human milk was associated with delayed time to rotavirus infection, implying protective properties of antirotavirus antibodies, whereas a higher IgA antibody concentration was associated with greater incidence of Campylobacter infection.CONCLUSIONThis comprehensive assessment of human milk antibody profiles may be used to guide the development of passive protection strategies against infant morbidity and mortality.FUNDINGBill and Melinda Gates Foundation grant OPP1172222 (to KMJ); Bill and Melinda Gates Foundation grant OPP1066764 funded the MDIG trial (to DER); University of Rochester CTSI and Environmental Health Sciences Center funded the Rochester Lifestyle study (to RJL); and R01 AI043596 funded PROVIDE (to WAP).

Environmental enteric dysfunction (EED) is a subclinical enteropathy challenging to diagnose due to an overlap of tissue features with other inflammatory enteropathies. EED subjects ( n  = 52) from Pakistan, controls ( n  = 25), and a validation EED cohort ( n  = 30) from Zambia were used to develop a machine-learning-based image analysis classification model. We extracted histologic feature representations from the Pakistan EED model and correlated them to transcriptomics and clinical biomarkers. In-silico metabolic network modeling was used to characterize alterations in metabolic flux between EED and controls and validated using untargeted lipidomics. Genes encoding beta-ureidopropionase, CYP4F3, and epoxide hydrolase 1 correlated to numerous tissue feature representations. Fatty acid and glycerophospholipid metabolism-related reactions showed altered flux. Increased phosphatidylcholine, lysophosphatidylcholine (LPC), and ether-linked LPCs, and decreased ester-linked LPCs were observed in the duodenal lipidome of Pakistan EED subjects, while plasma levels of glycine-conjugated bile acids were significantly increased. Together, these findings elucidate a multi-omic signature of EED., Competing Interests: KDRS has equity in Asklepion Pharmaceuticals and is a consultant to Travere Therapeutics and Mirum Pharmaceuticals. All the other authors have no conflicts of interest to disclose., (© 2024 The Authors.)

Background: Pakistan is endemic to a diverse set of parasitic, mycobacterial and viral diseases. The recognition of BCG Trained Immunity (TI) led us to postulate that the continued presence of BCG-TI may play a protective role, previously reported for both infectious and noninfectious conditions. Most of the previous studies have addressed the issue of BCG-TI in the paediatric populations. This study addressed the key issue of maintenance of BCG-TI in a wider age range (adolescent and adults) to identify the strength and quality of the immune responses., Objective: To assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections., Study Design: In this cross-sectional study, healthy young adults and adolescents (n = 20) were recruited from 16-40 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition., Methods: BCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network [macrophages [M1 (TNFα, IFNγ), M2 (IL10)], NK (IL2), Gamma delta (γδ) T (IL17, IL4)] and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay., Results: Significant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNFα (9148 pg/ml) followed by IL10 (488 pg/ml) and IFNγ (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNFα, 40 fold fo r IL10, and 27 fold for IFNγ. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG., Conclusion: The presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro- and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Iqbal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Protection against SARS-CoV-2 is mediated by humoral and T cell responses. Pakistan faced relatively low morbidity and mortality from COVID-19 through the pandemic. To examine the role of prior immunity in the population, we studied IgG antibody response levels, virus neutralizing activity and T cell reactivity to Spike protein in a healthy control group (HG) as compared with COVID-19 cases and individuals from the pre-pandemic period (PP)., Methods: HG and COVID-19 participants were recruited between October 2020 and May 2021. Pre-pandemic sera was collected before 2018. IgG antibodies against Spike and its Receptor Binding Domain (RBD) were determined by ELISA. Virus neutralization activity was determined using a PCR-based micro-neutralization assay. T cell - IFN-γ activation was assessed by ELISpot., Results: Overall, the magnitude of anti-Spike IgG antibody levels as well as seropositivity was greatest in COVID-19 cases (90%) as compared with HG (39.8%) and PP (12.2%). During the study period, Pakistan experienced three COVID-19 waves. We observed that IgG seropositivity to Spike in HG increased from 10.3 to 83.5% during the study, whilst seropositivity to RBD increased from 7.5 to 33.3%. IgG antibodies to Spike and RBD were correlated positively in all three study groups. Virus neutralizing activity was identified in sera of COVID-19, HG and PP. Spike reactive T cells were present in COVID-19, HG and PP groups. Individuals with reactive T cells included those with and without IgG antibodies to Spike., Conclusions: Antibody and T cell responses to Spike protein in individuals from the pre-pandemic period suggest prior immunity against SARS-CoV-2, most likely from cross-reactive responses. The rising seroprevalence observed in healthy individuals through the pandemic without known COVID-19 may be due to the activation of adaptive immunity from cross-reactive memory B and T cells. This may explain the more favourable COVID-19 outcomes observed in this population., (© 2023. The Author(s).)

The acquisition of antimicrobial resistance (AR) genes has rendered important pathogens nearly or fully unresponsive to antibiotics. It has been suggested that pathogens acquire AR traits from the gut microbiota, which collectively serve as a global reservoir for AR genes conferring resistance to all classes of antibiotics. However, only a subset of AR genes confers resistance to clinically relevant antibiotics, and, although these AR gene profiles are well-characterized for common pathogens, less is known about their taxonomic associations and transfer potential within diverse members of the gut microbiota. We examined a collection of 14,850 human metagenomes and 1666 environmental metagenomes from 33 countries, in addition to nearly 600,000 isolate genomes, to gain insight into the global prevalence and taxonomic range of clinically relevant AR genes. We find that several of the most concerning AR genes, such as those encoding the cephalosporinase CTX-M and carbapenemases KPC, IMP, NDM, and VIM, remain taxonomically restricted to Proteobacteria. Even cfiA, the most common carbapenemase gene within the human gut microbiome, remains tightly restricted to Bacteroides, despite being found on a mobilizable plasmid. We confirmed these findings in gut microbiome samples from India, Honduras, Pakistan, and Vietnam, using a high-sensitivity single-cell fusion PCR approach. Focusing on a set of genes encoding carbapenemases and cephalosporinases, thus far restricted to Bacteroides species, we find that few mutations are required for efficacy in a different phylum, raising the question of why these genes have not spread more widely. Overall, these data suggest that globally prevalent, clinically relevant AR genes have not yet established themselves across diverse commensal gut microbiota., (© 2023. The Author(s).)

Background: We previously reported unexpected Rh antibodies in the plasma of patients with sickle cell disease (SCD) that demonstrated common Rh specificities in the absence of transfusion of RBCs positive for that antigen. We hypothesize that these antibodies might result from transfusion of antigen-negative donor units with variant RH genotypes., Methods: Plasma testing by tube and IgG gel, extended RBC phenotyping, and HEA and RH genotyping were by standard methods., Case: A 6-year-old female with SCD, phenotype D + C-c + E-e + K- undergoing exchange transfusion with CEK- and Fy(a-) units, presented with anti-C in the plasma, a + DAT and warm autoantibody (WAA) in the eluate. Her RH genotype was unremarkable: RHD*D/DAU0 and RHCE*ce/ce(48C). Units (n = 10) transfused over the prior 6 months were confirmed CEK- by serology and DNA testing. Most (n = 7) were Rh-negative. A unit with variant RH, RHD*DIIIa/weak partial 4.0, RHCE*ceVS.03/ceVS.02, was transfused 5 weeks prior. Anti-C and + DAT continued to demonstrate for 25 weeks. Total hemoglobin and % Hgb S did not deviate from her established baseline., Conclusion: We show direct association of plasma anti-C with transfusion of a C-negative unit with variant RH encoding partial D and uncommon V/VS+ hr B - phenotype. The antibody was transient, without evidence of compromised survival of transfused RBCs. The +DAT and WAA complicated workups and selection of units, and it is uncertain whether donors of the same genotype should be avoided. Minority donors are important for CEK-matching to avoid depleting Rh-negative supplies. Consideration of patient and donor RH genotypes may avoid unexpected antibodies and improve allocation of rare donations., (© 2023 Wiley Periodicals LLC.)

Background: Diarrhoea and acute respiratory infections (ARI) are assumed to be major drivers of growth and likely contribute to environmental enteric dysfunction (EED), which is a precursor to childhood malnutrition. In the present study, we checked the correlation between diarrhoeal/ARI burden and EED using a novel duodenal histological index., Methods: Between November 2017 and July 2019, a total of 365 infants with weight-for-height Z scores (WHZ score) of <-2 were enrolled, and 51 infants with WHZ scores of >0 and height-for-age Z scores (HAZ scores) of >-1 were selected as age-matched healthy controls. Morbidity was assessed weekly and categorised as the total number of days with diarrhoea and acute respiratory infection (ARI) from enrolment until two years of age and was further divided into four quartiles in ascending order., Findings: The HAZ declined until two years of age regardless of morbidity burden, and WHZ and weight-for-age Z scores (WAZ scores) were at their lowest at six months. Sixty-three subjects who had a WHZ score <-2 and failed to respond to nutritional and educational interventions were further selected at 15 months to investigate their EED histological scores with endoscopy further. EED histological scores of the subjects were higher with increasing diarrhoeal frequency yet remained statistically insignificant (p = 0.810)., Interpretation: There was not a clear correlation between diarrhoea and ARI frequency with growth faltering, however, children with the highest frequency of diarrhoea had the highest EED histological scores and growth faltering., Funding: Bill and Melinda Gates Foundation and The National Institutes of Health., Competing Interests: This work was supported by the 10.13039/100000865Bill and Melinda Gates Foundation (grant number OPP1138727 to SAA and grant number OPP1144149 to SRM and The National Institutes of Health (grant number 2D43TW007585-2 to AA and SRM)., (© 2023 The Author(s).)

Objectives: Staphylococcus aureus and Streptococcus pneumoniae are common colonizers of the human nasopharynx. In this study, we describe S. aureus nasopharyngeal carriage and evaluate its association with S. pneumoniae carriage post-10-valent pneumococcal conjugate vaccine (PCV10) introduction in Pakistan., Methods: A serial cross-sectional study was undertaken from 2014 to 2018, children <2 years were randomly selected, and nasopharyngeal swabs were collected using standard WHO guidelines. S. aureus and S. pneumoniae isolates were identified using standard methods and tested for antimicrobial susceptibility by the standard Kirby-Bauer disk-diffusion method as per Clinical & Laboratory Standards Institute (CLSI) recommendations. Regression analysis was used to determine predictors associated with S. aureus carriage., Results: We enrolled 3140 children. S. aureus carriage prevalence was 5.6% (176/3140), and 50.1% (81/176) of the isolates were methicillin-resistant S. aureus (MRSA). S. aureus carriage was higher in the absence of pneumococcus compared to isolates in which pneumococcus was present (7.5% vs 5.0%). S. aureus carriage was negatively associated with pneumococcal carriage, being in 3rd and 4th year of enrollment, and vaccination with two and three PCV10 doses, in addition, fast breathing, ≥2 outpatients visits, and rainy season were positively associated. The following resistance rates were observed: 98.9% for penicillin, 74.4% for fusidic acid, and 23.3% for gentamicin, 10.2% for erythromycin, and 8.5% for cotrimoxazole. All isolates were susceptible to amikacin., Conclusions: Overall S. aureus carriage prevalence was low, PCV10 vaccine was protective against the carriage. The proportion of MRSA carriage and antimicrobial resistance was high in this community warranting continuous monitoring for invasive infections., Competing Interests: The authors have no relevant financial or non-financial interests to disclose., (© 2023 The Authors.)

Background: Pakistan has a well-established Expanded Program on Immunization (EPI) however vaccine-preventable diseases still account for high infant and child mortality rates. This study describes the differential vaccine coverage and determinants of vaccine uptake in rural Pakistan., Methods: From October 2014 to September 2018, we enrolled children younger than 2 years of age from the Matiari Demographic Surveillance System in Sindh, Pakistan. Socio-demographic and vaccination history were collected from all participants. Vaccine coverage rates and timeliness were reported. Socio-demographic variables for missed and untimely vaccination were studied in multivariable logistic regression., Results: Of the 3140 enrolled children, 48.4 % received all EPI recommended vaccines. Only 21.2 % of these were age appropriate. Around 45.4 % of the children were partially vaccinated, and 6.2 % were unvaccinated. Highest coverage was seen for the first dose of pentavalent (72.8 %), 10-valent Pneumococcal Conjugate Vaccine (PCV10) (70.4 %) and Oral Polio Vaccine (OPV) (69.2 %) and the lowest coverage was for measles (29.3 %) and rotavirus (1.8 %) vaccines. Primary caretakers and wage earners with a higher level of education were protective against missed and untimely vaccination. Enrollment in the 2nd, 3rd and 4th study year was negatively associated with being unvaccinated whereas distance from a major road was positively associated with non-adherence to schedule., Conclusion: Vaccine coverage was low among children in Matiari, Pakistan, and majority received delayed doses. Parents' education status and year of study enrollment was protective against vaccine dropout and delayed vaccination whereas geographical distance from a major road was a predictor. Vaccine promotion and outreach efforts may have had a beneficial impact on vaccine coverage and timeliness., 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 © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Environmental enteric dysfunction (EED) is characterized by malabsorption and diarrhea that result in irreversible deficits in physical and intellectual growth. We sought to define the expression of transport and tight junction proteins by quantitative analysis of duodenal biopsies from patients with EED. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age-matched North American healthy controls, patients with celiac disease, and patients with nonceliac disease with villous atrophy or intraepithelial lymphocytosis. Expression of brush border digestive and transport proteins and paracellular (tight junction) proteins was assessed by quantitative multiplex immunofluorescence microscopy. EED was characterized by partial villous atrophy and marked intraepithelial lymphocytosis. Epithelial proliferation and enteroendocrine, tuft, and Paneth cell numbers were unchanged, but there was significant goblet cell expansion in EED biopsies. Expression of proteins involved in nutrient and water absorption and that of the basolateral Cl - transport protein NKCC1 were also increased in EED. Finally, the barrier-forming tight junction protein claudin-4 (CLDN4) was significantly upregulated in EED, particularly within villous enterocytes. In contrast, expression of CFTR, CLDN2, CLDN15, JAM-A, occludin, ZO-1, and E-cadherin was unchanged. Upregulation of a barrier-forming tight junction protein and brush border and basolateral membrane proteins that support nutrient and water transport in EED is paradoxical, as their increased expression would be expected to be correlated with increased intestinal barrier function and enhanced absorption, respectively. These data suggest that EED activates adaptive intestinal epithelial responses to enhance nutrient absorption but that these changes are insufficient to restore health., (Copyright © 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Environmental enteric dysfunction (EED) is characterized by intestinal inflammation, malabsorption and growth-faltering in children with heightened exposure to gut pathogens. The aim of this study was to characterize serum non-esterified fatty acids (NEFA), in association with childhood undernutrition and EED, as potential biomarkers to predict growth outcomes. The study comprised a cohort of undernourished rural Pakistani infants ( n = 365) and age-matched controls followed prospectively up to 24 months of age. Serum NEFA were quantified at ages 3-6 and 9 months and correlated with growth outcomes, serum bile acids and EED histopathological biomarkers. Serum NEFA correlated with linear growth-faltering and systemic and gut biomarkers of EED. Undernourished children exhibited essential fatty acid deficiency (EFAD), with low levels of linoleic acid and total n-6 polyunsaturated fatty acids, compensated by increased levels of oleic acid and increased elongase and desaturase activities. EFAD correlated with reduced anthropometric Z scores at 3-6 and 9 months of age. Serum NEFA also correlated with elevated BA and liver dysfunction. Essential fatty acid depletion and altered NEFA metabolism were highly prevalent and associated with acute and chronic growth-faltering in EED. The finding suggests that targeting early interventions to correct EFAD and promote FA absorption in children with EED may facilitate childhood growth in high-risk settings.

Introduction: Environmental enteropathy is an important contributor to childhood malnutrition in the developing world. Chronic exposure to fecal pathogens leads to alteration in intestinal structure and function, resulting in impaired gut immune function, malabsorption, and growth faltering leading to environmental enteropathy., Methods: A community-based intervention study was carried out on children till 24 months of age in Matiari district, Pakistan. Blood and fecal specimens were collected from the enrolled children aged 3-6 and 9 months. A real-time PCR-based TaqMan array card (TAC) was used to detect enteropathogens., Results: Giardia , Campylobacter spp., enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Cryptosporidium spp. were the most prevailing enteropathogens in terms of overall positivity at both time points. Detection of protozoa at enrollment and 9 months was negatively correlated with rate of change in height-for-age Z (ΔHAZ) scores during the first and second years of life. A positive association was found between Giardia, fecal lipocalin (LCN), and alpha 1-Acid Glycoprotein (AGP), while Campylobacter spp. showed positive associations with neopterin (NEO) and myeloperoxidase (MPO)., Conclusion: Protozoal colonization is associated with a decline in linear growth velocity during the first 2 years of life in children living in Environmental enteric dysfunction (EED) endemic settings. Mechanistic studies exploring the role of cumulative microbial colonization, their adaptations to undernutrition, and their influence on gut homeostasis are required to understand symptomatic enteropathogen-induced growth faltering., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kabir, Iqbal, Jamil, Iqbal, Mallawaarachchi, Aziz, Kalam, Muneer, Hotwani, Ahmed, Umrani, Syed, Sadiq, Ma, Moore and Ali.)

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S 1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S 1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S 1 -directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S 1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.

Background: Early childhood caries poses a significant health issue in children under 6 years old. It is determined that Streptococcus mutans is a primary etiological agent, likely to be transferred through maternal contact., Objectives: To determine the association of maternal S. mutans counts with S. mutans counts in their children between 6 and 30 months of age, and to determine the maternal and child DMFT (decayed, missing, and filled teeth) indices., Material and Methods: A community-based cross-sectional study was conducted in Karachi, Pakistan. A sample of 193 dyads of mother-children (6-30 months of age) was selected via purposive sampling. Saliva samples of the dyads were collected to assess S. mutans count. Caries assessment was performed for both using the DMFT index. A pretested questionnaire was used. The association of bottle-feeding, oral hygiene measures, and other factors with S. mutans counts in children were also explored. Zero-inflated negative binomial regression model at a 5% level of significance was applied using STATA version 12.0., Results: Out of 193 children, 109 (56.47%) were males and 84 (43.52%) were females. The mean age of mothers and children was 29.4 ± 6.2 years and 19.54 ± 6.8 months, respectively. Maternal S. mutans counts were not statistically associated with child's S. mutans counts (Mean child's S. mutans count ratio: 1; 95% confidence interval [CI]: 1, 1.01; p = .882). Compared with children who were breastfed, S. mutans counts were higher in children who were bottle-fed (mean S. mutans count ratio= 4.85 [95% CI: 1.53, 15.41], p = .007). Age of mother and present caries status of mothers was significantly associated with the child's S. mutans count., Conclusion: No association between maternal S. mutans and child S. mutans was observed. However, maternal age, children who were breastfed, children who did not use pacifiers, and children with mothers who did not have caries, exhibited low S. mutans counts in their saliva., (© 2022 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern comprises several sublineages, with BA.2 and BA.2.12.1 having replaced the previously dominant BA.1 and with BA.4 and BA.5 increasing in prevalence worldwide. We show that the large number of Omicron sublineage spike mutations leads to enhanced angiotensin-converting enzyme 2 (ACE2) binding, reduced fusogenicity, and severe dampening of plasma neutralizing activity elicited by infection or seven clinical vaccines relative to the ancestral virus. Administration of a homologous or heterologous booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 across all vaccines evaluated. Our data suggest that although Omicron sublineages evade polyclonal neutralizing antibody responses elicited by primary vaccine series, vaccine boosters may provide sufficient protection against Omicron-induced severe disease.

Stunting is a global public health issue. We sought to train and evaluate machine learning (ML) classification algorithms on the Zambia Demographic Health Survey (ZDHS) dataset to predict stunting among children under the age of five in Zambia. We applied Logistic regression (LR), Random Forest (RF), SV classification (SVC), XG Boost (XgB) and Naïve Bayes (NB) algorithms to predict the probability of stunting among children under five years of age, on the 2018 ZDHS dataset. We calibrated predicted probabilities and plotted the calibration curves to compare model performance. We computed accuracy, recall, precision and F1 for each machine learning algorithm. About 2327 (34.2%) children were stunted. Thirteen of fifty-eight features were selected for inclusion in the model using random forest. Calibrating the predicted probabilities improved the performance of machine learning algorithms when evaluated using calibration curves. RF was the most accurate algorithm, with an accuracy score of 79% in the testing and 61.6% in the training data while Naïve Bayesian was the worst performing algorithm for predicting stunting among children under five in Zambia using the 2018 ZDHS dataset. ML models aids quick diagnosis of stunting and the timely development of interventions aimed at preventing stunting.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to acute respiratory distress syndrome (ARDS). Understanding the evolution of the virus, and immune-pathogenic processes are critical for designing future therapeutic interventions. In this review, we collate information on the structure, genome, viral life cycle, and adult and pediatric host immune responses in response to SARS-CoV-2. The immunological responses are a prototype of the developmental origins of health and disease (DOHaD) hypothesis to explain the socio-geographic differences impacting the severity and mortality rates in SARS-CoV-2 infections. The DOHaD hypothesis identifies the relevance of trained innate immunity, age groups, and geography for effective vaccinations. As COVID-19 vaccines are being rolled out, it may be pertinent to assess population-based immunological responses to understand the effectiveness and safety across different populations and age groups.

Bifidobacterium longum subspecies detected in infant stool have been associated with numerous subsequent health outcomes and are potential early markers of deviation from healthy developmental trajectories. This analysis derived indicators of carriage and early colonization with B. infantis and B. longum and quantified their associations with a panel of early-life exposures and outcomes. In a sub-study nested within a multi-site birth cohort, extant stool samples from infants in Bangladesh, Pakistan and Tanzania were tested for presence and quantity of two Bifidobacterium longum subspecies. The results were matched to indicators of nutritional status, enteropathogen infection, histo-blood group antigens, vaccine response and feeding status and regression models were fitted to test for associations while adjusting for covariates. B. infantis was associated with lower quantity of and decreased odds of colonization with B. longum , and vice versa. Length at birth was associated with a 0.36 increase in log 10 B. infantis and a 0.28 decrease in B. longum quantity at 1 month of age. B. infantis colonization was associated with fewer viral infections and small reductions in the risk of rotavirus and sapovirus infections, but not reduced overall diarrheal disease risk. No associations with vaccine responses, HBGAs or later nutritional status were identified. Suboptimal intrauterine growth and a shorter duration of exclusive breastfeeding may predispose infants to early intestinal colonization with the B. longum subspecies at the expense of B. infantis , thus denying them potential benefits of reduced enteric virus episodes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Colston, Taniuchi, Ahmed, Ferdousi, Kabir, Mduma, Nshama, Iqbal, Haque, Ahmed, Ali Bhutta, Kosek and Platts-Mills.)

The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody 1 . Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab 2 , S2X259 3 and S2H97 4 . The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Intestinal inflammation and malabsorption in environmental enteric dysfunction (EED) are associated with early childhood growth faltering in impoverished settings worldwide., Objectives: The goal of this study was to identify candidate biomarkers associated with inflammation, EED histology, and as predictors of later growth outcomes by focusing on the liver-gut axis by investigating the bile acid metabolome., Methods: Undernourished rural Pakistani infants (n = 365) with weight-for-height Z score (WHZ) < -2 were followed up to the age of 24 mo and monitored for growth, infections, and EED. Well-nourished local children (n = 51) were controls, based on consistent WHZ > 0 and height-for-age Z score (HAZ) > -1 on 2 consecutive visits at 3 and 6 mo. Serum bile acid (sBA) profiles were measured by tandem MS at the ages of 3-6 and 9 mo and before nutritional intervention. Biopsies and duodenal aspirates were obtained following upper gastrointestinal endoscopy from a subset of children (n = 63) that responded poorly to nutritional intervention. BA composition in paired plasma and duodenal aspirates was compared based on the severity of EED histopathological scores and correlated to clinical and growth outcomes., Results: Remarkably, >70% of undernourished Pakistani infants displayed elevated sBA concentrations consistent with subclinical cholestasis. Serum glycocholic acid (GCA) correlated with linear growth faltering (HAZ, r = -0.252 and -0.295 at the age of 3-6 and 9 mo, respectively, P <0.001) and biomarkers of inflammation. The proportion of GCA positively correlated with EED severity for both plasma (rs = 0.324 P = 0.02) and duodenal aspirates (rs = 0.307 P = 0.06) in children with refractory wasting that underwent endoscopy, and the proportion of secondary BA was low in both undernourished and EED children., Conclusions: Dysregulated bile acid metabolism is associated with growth faltering and EED severity in undernourished children. Restoration of intestinal BA homeostasis may offer a novel therapeutic target for undernutrition in children with EED. This trial was registered at clinicaltrials.gov as NCT03588013., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

The relationship between environmental factors and child health is not well understood in rural Pakistan. This study characterized the environmental factors related to the morbidity of acute respiratory infections (ARIs), diarrhea, and growth using geographical information systems (GIS) technology. Anthropometric, address and disease prevalence data were collected through the SEEM (Study of Environmental Enteropathy and Malnutrition) study in Matiari, Pakistan. Publicly available map data were used to compile coordinates of healthcare facilities. A Pearson correlation coefficient ( r ) was used to calculate the correlation between distance from healthcare facilities and participant growth and morbidity. Other continuous variables influencing these outcomes were analyzed using a random forest regression model. In this study of 416 children, we found that participants living closer to secondary hospitals had a lower prevalence of ARI ( r = 0.154, p < 0.010) and diarrhea (r = 0.228, p < 0.001) as well as participants living closer to Maternal Health Centers (MHCs): ARI ( r = 0.185, p < 0.002) and diarrhea ( r = 0.223, p < 0.001) compared to those living near primary facilities. Our random forest model showed that distance has high variable importance in the context of disease prevalence. Our results indicated that participants closer to more basic healthcare facilities reported a higher prevalence of both diarrhea and ARI than those near more urban facilities, highlighting potential public policy gaps in ameliorating rural health.

Background: Impaired growth among infants remains one of the leading nutrition problems globally. In this study, we aimed to compare the growth trajectory rate and evaluate growth trajectory characteristics among children, who are HIV exposed uninfected (HEU) and HIV unexposed uninfected (HUU), under two years in Zambia., Method: Our study used data from the ROVAS II study (PACTR201804003096919), an open-label randomized control trial of two verses three doses of live, attenuated, oral RotarixTM administered 6 &10 weeks or at 6 &10 weeks plus an additional dose at 9 months of age, conducted at George clinic in Lusaka, Zambia. Anthropometric measurements (height and weight) were collected on all scheduled and unscheduled visits. We defined linear growth velocity as the rate of change in height and estimated linear growth velocity as the first derivative of the mixed effect model with fractional polynomial transformations and, thereafter, used the second derivative test to determine the peak height and age at peak heigh., Results: We included 212 infants in this study with median age 6 (IQR: 6-6) weeks of age. Of these 97 (45.3%) were female, 35 (16.4%) were stunted, and 59 (27.6%) were exposed to HIV at baseline. Growth velocity was consistently below the 3rd percentile of the WHO linear growth standard for HEU and HUU children. The peak height and age at peak height among HEU children were 74.7 cm (95% CI = 73.9-75.5) and 15.5 months (95% CI = 14.7-16.3) respectively and those for HUU were 73 cm (95% CI = 72.1-74.0) and 15.6 months (95% CI = 14.5-16.6) respectively., Conclusion: We found no difference in growth trajectories between infants who are HEU and HUU. However, the data suggests that poor linear growth is universal and profound in this cohort and may have already occurred in utero., Competing Interests: The authors have declared that no competing interests exist.

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.)

Environmental enteric dysfunction (EED) is a subclinical condition of intestinal inflammation, barrier dysfunction and malabsorption associated with growth faltering in children living in poverty. This study explores association of altered duodenal permeability (lactulose, rhamnose and their ratio) with higher burden of enteropathogen in the duodenal aspirate, altered histopathological findings and higher morbidity (diarrhea) that is collectively associated with linear growth faltering in children living in EED endemic setting. In a longitudinal birth cohort, 51 controls (WHZ > 0, HAZ > -1.0) and 63 cases (WHZ< -2.0, refractory to nutritional intervention) were recruited. Anthropometry and morbidity were recorded on monthly bases up to 24 months of age. Dual sugar assay of urine collected after oral administration of lactulose and rhamnose was assessed in 96 children from both the groups. Duodenal histopathology (n = 63) and enteropathogen analysis of aspirate via Taqman array card (n = 60) was assessed in only cases. Giardia was the most frequent pathogen and was associated with raised L:R ratio (p = 0.068). Gastric microscopy was more sensitive than duodenal aspirate in H. pylori detection. Microscopically confirmed H. pylori negatively correlated with HAZ at 24 months (r = -0.313, p = 0.013). Regarding histopathological parameters, goblet cell reduction significantly correlated with decline in dual sugar excretion (p< 0.05). Between cases and controls, there were no significant differences in the median (25th, 75th percentile) of urinary concentrations (μg/ml) of lactulose [27.0 (11.50, 59.50) for cases vs. 38.0 (12.0, 61.0) for controls], rhamnose [66.0 (28.0, 178.0) vs. 86.5 (29.5, 190.5)] and L:R ratio [0.47 (0.24, 0.90) vs. 0.51 (0.31, 0.71)] respectively. In multivariable regression model, 31% of variability in HAZ at 24 months of age among cases and controls was explained by final model including dual sugars. In conclusion, enteropathogen burden is associated with altered histopathological features and intestinal permeability. In cases and controls living in settings of endemic enteropathy, intestinal permeability test may predict linear growth. However, for adoption as a screening tool for EED, further validation is required due to its complex intestinal pathophysiology., Competing Interests: The authors have declared that no competing interests exist.

Background: Prolonged enteropathogen shedding after diarrhea complicates the identification of etiology in subsequent episodes and is an important driver of pathogen transmission. A standardized approach has not been applied to estimate the duration of shedding for a wide range of pathogens., Methods: We used a multisite birth cohort of children 0-24 months of age from whom diarrheal and monthly nondiarrheal stools were previously tested by quantitative polymerase chain reaction for 29 enteropathogens. We modeled the probability of detection of the etiologic pathogen before and after diarrhea using a log-normal accelerated failure time survival model and estimated the median duration of pathogen carriage as well as differences in subclinical pathogen carriage 60 days after diarrhea onset in comparison to a prediarrhea baseline., Results: We analyzed 3247 etiologic episodes of diarrhea for the 9 pathogens with the highest attributable burdens of diarrhea. The median duration of postdiarrheal carriage varied widely by pathogen, from about 1 week for rotavirus (median, 8.1 days [95% confidence interval {CI}, 6.2-9.6]) to >1 month for Cryptosporidium (39.5 days [95% CI, 30.6-49.0]). The largest increases in subclinical pathogen carriage before and after diarrhea were seen for Cryptosporidium (prevalence difference between 30 days prior and 60 days after diarrhea onset, 0.30 [95% CI, .23-.39]) and Shigella (prevalence difference, 0.21 [95% CI, .16-.27])., Conclusions: Postdiarrheal shedding was widely variable between pathogens, with strikingly prolonged shedding seen for Cryptosporidium and Shigella. Targeted antimicrobial therapy and vaccination for these pathogens may have a relatively large impact on transmission., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objectives: Striking histopathological overlap between distinct but related conditions poses a disease diagnostic challenge. There is a major clinical need to develop computational methods enabling clinicians to translate heterogeneous biomedical images into accurate and quantitative diagnostics. This need is particularly salient with small bowel enteropathies; environmental enteropathy (EE) and celiac disease (CD). We built upon our preliminary analysis by developing an artificial intelligence (AI)-based image analysis platform utilizing deep learning convolutional neural networks (CNNs) for these enteropathies., Methods: Data for the secondary analysis was obtained from three primary studies at different sites. The image analysis platform for EE and CD was developed using CNNs including one with multizoom architecture. Gradient-weighted class activation mappings (Grad-CAMs) were used to visualize the models' decision-making process for classifying each disease. A team of medical experts simultaneously reviewed the stain color normalized images done for bias reduction and Grad-CAMs to confirm structural preservation and biomedical relevance, respectively., Results: Four hundred and sixty-one high-resolution biopsy images from 150 children were acquired. Median age (interquartile range) was 37.5 (19.0-121.5) months with a roughly equal sex distribution; 77 males (51.3%). ResNet50 and shallow CNN demonstrated 98% and 96% case-detection accuracy, respectively, which increased to 98.3% with an ensemble. Grad-CAMs demonstrated models' ability to learn different microscopic morphological features for EE, CD, and controls., Conclusions: Our AI-based image analysis platform demonstrated high classification accuracy for small bowel enteropathies which was capable of identifying biologically relevant microscopic features and emulating human pathologist decision-making process. Grad-CAMs illuminated the otherwise "black box" of deep learning in medicine, allowing for increased physician confidence in adopting these new technologies in clinical practice., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Background & Aims: Environmental enteric dysfunction (EED) limits the Sustainable Development Goals of improved childhood growth and survival. We applied mucosal genomics to advance our understanding of EED., Methods: The Study of Environmental Enteropathy and Malnutrition (SEEM) followed 416 children from birth to 24 months in a rural district in Pakistan. Biomarkers were measured at 9 months and tested for association with growth at 24 months. The duodenal methylome and transcriptome were determined in 52 undernourished SEEM participants and 42 North American controls and patients with celiac disease., Results: After accounting for growth at study entry, circulating insulin-like growth factor-1 (IGF-1) and ferritin predicted linear growth, whereas leptin correlated with future weight gain. The EED transcriptome exhibited suppression of antioxidant, detoxification, and lipid metabolism genes, and induction of anti-microbial response, interferon, and lymphocyte activation genes. Relative to celiac disease, suppression of antioxidant and detoxification genes and induction of antimicrobial response genes were EED-specific. At the epigenetic level, EED showed hyper-methylation of epithelial metabolism and barrier function genes, and hypo-methylation of immune response and cell proliferation genes. Duodenal coexpression modules showed association between lymphocyte proliferation and epithelial metabolic genes and histologic severity, fecal energy loss, and wasting (weight-for-length/height Z < -2.0). Leptin was associated with expression of epithelial carbohydrate metabolism and stem cell renewal genes. Immune response genes were attenuated by giardia colonization., Conclusions: Children with reduced circulating IGF-1 are more likely to experience stunting. Leptin and a gene signature for lymphocyte activation and dysregulated lipid metabolism are implicated in wasting, suggesting new approaches for EED refractory to nutritional intervention. ClinicalTrials.gov, Number: NCT03588013. (https://clinicaltrials.gov/ct2/show/NCT03588013)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Stunting affects up to one-third of the children in low-to-middle income countries (LMICs) and has been correlated with decline in cognitive capacity and vaccine immunogenicity. Early identification of infants at risk is critical for early intervention and prevention of morbidity. The aim of this study was to investigate patterns of growth in infants up through 48 months of age to assess whether the growth of infants with stunting eventually improved as well as the potential predictors of growth., Methods: Height-for-age z-scores (HAZ) of children from Matiari (rural site, Pakistan) at birth, 18 months, and 48 months were obtained. Results of serum-based biomarkers collected at 6 and 9 months were recorded. A descriptive analysis of the population was followed by assessment of growth predictors via traditional machine learning random forest models., Results: Of the 107 children who were followed up till 48 months of age, 51% were stunted (HAZ < - 2) at birth which increased to 54% by 48 months of age. Stunting status for the majority of children at 48 months was found to be the same as at 18 months. Most children with large gains started off stunted or severely stunted, while all of those with notably large losses were not stunted at birth. Random forest models identified HAZ at birth as the most important feature in predicting HAZ at 18 months. Of the biomarkers, AGP (Alpha- 1-acid Glycoprotein), CRP (C-Reactive Protein), and IL1 (interleukin-1) were identified as strong subsequent growth predictors across both the classification and regressor models., Conclusion: We demonstrated that children most children with stunting at birth remained stunted at 48 months of age. Value was added for predicting growth outcomes with the use of traditional machine learning random forest models. HAZ at birth was found to be a strong predictor of subsequent growth in infants up through 48 months of age. Biomarkers of systemic inflammation, AGP, CRP, IL1, were also strong predictors of growth outcomes. These findings provide support for continued focus on interventions prenatally, at birth, and early infancy in children at risk for stunting who live in resource-constrained regions of the world.

Culture-independent diagnostics have revealed a larger burden of Shigella among children in low-resource settings than previously recognized. We further characterized the epidemiology of Shigella in the first two years of life in a multisite birth cohort. We tested 41,405 diarrheal and monthly non-diarrheal stools from 1,715 children for Shigella by quantitative PCR. To assess risk factors, clinical factors related to age and culture positivity, and associations with inflammatory biomarkers, we used log-binomial regression with generalized estimating equations. The prevalence of Shigella varied from 4.9%-17.8% in non-diarrheal stools across sites, and the incidence of Shigella-attributable diarrhea was 31.8 cases (95% CI: 29.6, 34.2) per 100 child-years. The sensitivity of culture compared to qPCR was 6.6% and increased to 27.8% in Shigella-attributable dysentery. Shigella diarrhea episodes were more likely to be severe and less likely to be culture positive in younger children. Older age (RR: 1.75, 95% CI: 1.70, 1.81 per 6-month increase in age), unimproved sanitation (RR: 1.15, 95% CI: 1.03, 1.29), low maternal education (<10 years, RR: 1.14, 95% CI: 1.03, 1.26), initiating complementary foods before 3 months (RR: 1.10, 95% CI: 1.01, 1.20), and malnutrition (RR: 0.91, 95% CI: 0.88, 0.95 per unit increase in weight-for-age z-score) were risk factors for Shigella. There was a linear dose-response between Shigella quantity and myeloperoxidase concentrations. The burden of Shigella varied widely across sites, but uniformly increased through the second year of life and was associated with intestinal inflammation. Culture missed most clinically relevant cases of severe diarrhea and dysentery., Competing Interests: The authors have declared that no competing interests exist.

Objective: To explore the link between altered thyroid profile and oxidative stress marker in females with unexplained infertility., Methods: The cross-sectional case-control study was carried out at the Islamabad Clinic Serving Infertile Couples, Islamabad, Pakistan, from June 2016 to August 2017, and comprised women aged 18-40 years regardless of ethnic background who were divided into two groups; those with unexplained infertility were the cases, while fertile women acted as the controls. Serum was analysed for triiodothyronine, thyroxine and thyroid stimulating hormone as well as for oxidative stress markers including manganese superoxide dismutase, glutathione reductase and adrenaline using enzymelinked immunosorbent assay. Data was analysed using SPSS 19., Results: Of the 88 subjects, there were 44(50%) in each of the two groups. There was no significant difference in terms of thyroids markers except thyroxine and thyroid stimulating hormone (p<0.05). There were significant differences in terms of oxidative stress markers between the groups (p<0.05). A significant positive correlation of thyroid stimulating hormone was observed with manganese superoxide dismutase and adrenaline (p<0.05) with a weak non significant association of glutathione reductase (p>0.05)., Conclusions: Increased thyroxine levels in females with unexplained infertility was associated with decrease in the serum levels of antioxidants.

Reliance on microbiologic methods to diagnose Mycobacterium tuberculosis infection is a suboptimal approach for children due in part to the paucibacillary nature of the disease. A blood-based biomarker assay, such as the mycobacterial-antibody-secreting cell (MASC) assay, could be a major advance for the field of study of pediatric tuberculosis (TB). Children <15 years of age with clinical concern for TB and age-matched children with no concern for TB were enrolled from outpatient clinics in Karachi, Pakistan. MASC, ferritin, and C-reactive protein (CRP) assays were performed, and results were compared among cases and controls, as well as among children with a case definition of "confirmed TB," "probable TB," or "possible TB." MASC responses were significantly higher among children with TB than among controls (0.41 optical density [OD] versus 0.28 OD, respectively, P  < 0.001), and the differences were largely driven by the data from children with confirmed TB ( P  = 0.002). Ferritin and CRP values were significantly higher among those with confirmed TB than among those with the other disease states and controls ( P  = 0.004 and P  = 0.019, respectively). The use of the MASC assay as a blood-based biomarker for TB disease shows some promise among children with microbiologically confirmed disease; however, the performance characteristics for the majority of young children with unconfirmed TB were suboptimal in this cohort. IMPORTANCE Tuberculosis (TB) in children represents a missed opportunity for diagnosis and preventive therapy. The magnitude or burden of disease in children is not fully understood due to our limitations with respect to exploring sensitive diagnostic algorithms. In a setting of TB endemicity in Pakistan, we carried out a proof-of-concept study to evaluate for the first time the performance of B cell analyses by the use of well-defined diagnostic criteria and NIH consensus guidelines as "culture-confirmed," "probable," and "possible" TB groups. In contrast to detection of serum antibody, we focused on mycobacterial-antibody-secreting cell (MASC) detection as a marker of active disease in children with a strong suspicion of TB. Further work exploring a larger panel of inflammatory biomarkers and enrichment of B cells with the objective of increasing the sensitivity of the current MASC assay would lead to the development of a field-friendly assay for timely diagnosis of childhood TB., (Copyright © 2020 Iqbal et al.)

Epsilon gamma delta beta (εγδβ) 0 - thalassemia is a very rare disorder that results from large deletions in the β-globin gene cluster which abolish all regional globin chain gene expression from that allele. Since it is an exceedingly rare cause of neonatal anemia and is not detected by routine newborn screening, it is usually not suspected clinically and commonly undiagnosed or misdiagnosed. In this study, we describe two patients diagnosed in our hospital with (εγδβ) 0 -thalassemia based on the results obtained from DNA microarray analysis of their peripheral blood. The first patient of mixed European descent presented as a neonate with microcytic hemolytic anemia, hyperbilirubinemia, hypoglycemia and hypothermia, and was found to have a 2.2 Mb loss that included the entire β-globin gene cluster and the locus control region (LCR). The second patient, also of mixed European descent, presented in the neonatal period with anemia, thrombocytopenia and cutaneous extramedullary hematopoiesis, and was found to have a 59 kb loss that included the β-globin LCR, HBE1, HBG1, and HBG2 genes. Both cases highlight the importance of recognizing the clinical features of (εγδβ) 0 -thalassemia and implementing appropriate testing to clarify the diagnosis and manage the condition., (Copyright © 2019. Published by Elsevier GmbH.)

Background: Few studies have evaluated the association between preexisting vitamin D deficiency and incident tuberculosis (TB). We assessed the impact of baseline vitamins D levels on TB disease risk., Methods and Findings: We assessed the association between baseline vitamin D and incident TB in a prospective cohort of 6,751 HIV-negative household contacts of TB patients enrolled between September 1, 2009, and August 29, 2012, in Lima, Peru. We screened for TB disease at 2, 6, and 12 months after enrollment. We defined cases as household contacts who developed TB disease at least 15 days after enrollment of the index patient. For each case, we randomly selected four controls from among contacts who did not develop TB disease, matching on gender and year of age. We also conducted a one-stage individual-participant data (IPD) meta-analysis searching PubMed and Embase to identify prospective studies of vitamin D and TB disease until June 8, 2019. We included studies that assessed vitamin D before TB diagnosis. In the primary analysis, we defined vitamin D deficiency as 25-(OH)D < 50 nmol/L, insufficiency as 50-75 nmol/L, and sufficiency as >75nmol/L. We estimated the association between baseline vitamin D status and incident TB using conditional logistic regression in the Lima cohort and generalized linear mixed models in the meta-analysis. We further defined severe vitamin D deficiency as 25-(OH)D < 25 nmol/L and performed stratified analyses by HIV status in the IPD meta-analysis. In the Lima cohort, we analyzed 180 cases and 709 matched controls. The adjusted odds ratio (aOR) for TB risk among participants with baseline vitamin D deficiency compared to sufficient vitamin D was 1.63 (95% CI 0.75-3.52; p = 0.22). We included seven published studies in the meta-analysis and analyzed 3,544 participants. In the pooled analysis, the aOR was 1.48 (95% CI 1.04-2.10; p = 0.03). The aOR for severe vitamin D deficiency was 2.05 (95% CI 0.87-4.87; p trend for decreasing 25-(OH)D levels from sufficient vitamin D to severe deficiency = 0.02). Among 1,576 HIV-positive patients, vitamin D deficiency conferred a 2-fold (aOR 2.18, 95% CI 1.22-3.90; p = 0.01) increased risk of TB, and the aOR for severe vitamin D deficiency compared to sufficient vitamin D was 4.28 (95% CI 0.85-21.45; p = 0.08). Our Lima cohort study is limited by the short duration of follow-up, and the IPD meta-analysis is limited by the number of possible confounding covariates available across all studies., Conclusion: Our findings suggest vitamin D predicts TB disease risk in a dose-dependent manner and that the risk of TB disease is highest among HIV-positive individuals with severe vitamin D deficiency. Randomized control trials are needed to evaluate the possible role of vitamin D supplementation on reducing TB disease risk., Competing Interests: The authors of this manuscript have the following competing interests: MBM is a member of the Editorial Board of PLOS Medicine. All other authors have declared no competing interests.

Environmental Enteric Dysfunction (EED) is an acquired small intestinal inflammatory condition underlying high rates of stunting in children <5 years of age in low- and middle-income countries. Children with EED are known to have repeated exposures to enteropathogens and environmental toxins that leads to malabsorptive syndrome. We aimed to characterize association of linear growth faltering with enteropathogen burden and subsequent changes in EED biomarkers. In a longitudinal birth cohort (n = 272), monthly anthropometric measurements (Length for Age Z score- LAZ) of asymptomatic children were obtained up to 18 months. Biological samples were collected at 6 and 9 months for the assessment of biomarkers. A customized TaqMan array card was used to target 40 enteropathogens in fecal samples. Linear regression was applied to study the effect of specific enteropathogen infection on change in linear growth (ΔLAZ). Presence of any pathogen in fecal sample correlated with serum flagellin IgA (6 mo, r = 0.19, p = 0.002), fecal Reg 1b (6 mo, r = 0.16, p = 0.01; 9mo, r = 0.16, p = 0.008) and serum Reg 1b (6 mo, r = 0.26, p<0.0001; 9 mo, r = 0.15, p = 0.008). At 6 months, presence of Campylobacter [β (SE) 7751.2 (2608.5), p = 0.003] and ETEC LT [β (SE) 7089.2 (3015.04), p = 0.019] was associated with increase in MPO. Giardia was associated with increase in Reg1b [β (SE) 72.189 (26.394), p = 0.006] and anti-flic IgA[β (SE) 0.054 (0.021), p = 0.0091]. Multiple enteropathogen infections in early life negatively correlated with ΔLAZ, and simultaneous changes in gut inflammatory and permeability markers. A combination vaccine targeting enteropathogens in early life could help in the prevention of future stunting., Competing Interests: The authors have declared that no competing interests exist.

Background: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority., Methods: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community., Discussion: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals., Trial Registration: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Importance: Duodenal biopsies from children with enteropathies associated with undernutrition, such as environmental enteropathy (EE) and celiac disease (CD), display significant histopathological overlap., Objective: To develop a convolutional neural network (CNN) to enhance the detection of pathologic morphological features in diseased vs healthy duodenal tissue., Design, Setting, and Participants: In this prospective diagnostic study, a CNN consisting of 4 convolutions, 1 fully connected layer, and 1 softmax layer was trained on duodenal biopsy images. Data were provided by 3 sites: Aga Khan University Hospital, Karachi, Pakistan; University Teaching Hospital, Lusaka, Zambia; and University of Virginia, Charlottesville. Duodenal biopsy slides from 102 children (10 with EE from Aga Khan University Hospital, 16 with EE from University Teaching Hospital, 34 with CD from University of Virginia, and 42 with no disease from University of Virginia) were converted into 3118 images. The CNN was designed and analyzed at the University of Virginia. The data were collected, prepared, and analyzed between November 2017 and February 2018., Main Outcomes and Measures: Classification accuracy of the CNN per image and per case and incorrect classification rate identified by aggregated 10-fold cross-validation confusion/error matrices of CNN models., Results: Overall, 102 children participated in this study, with a median (interquartile range) age of 31.0 (20.3-75.5) months and a roughly equal sex distribution, with 53 boys (51.9%). The model demonstrated 93.4% case-detection accuracy and had a false-negative rate of 2.4%. Confusion metrics indicated most incorrect classifications were between patients with CD and healthy patients. Feature map activations were visualized and learned distinctive patterns, including microlevel features in duodenal tissues, such as alterations in secretory cell populations., Conclusions and Relevance: A machine learning-based histopathological analysis model demonstrating 93.4% classification accuracy was developed for identifying and differentiating between duodenal biopsies from children with EE and CD. The combination of the CNN with a deconvolutional network enabled feature recognition and highlighted secretory cells' role in the model's ability to differentiate between these histologically similar diseases.

Background: Enteropathogen infections in early childhood not only cause diarrhoea but contribute to poor growth. We used molecular diagnostics to assess whether particular enteropathogens were associated with linear growth across seven low-resource settings., Methods: We used quantitative PCR to detect 29 enteropathogens in diarrhoeal and non-diarrhoeal stools collected from children in the first 2 years of life obtained during the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) multisite cohort study. Length was measured monthly. We estimated associations between aetiology-specific diarrhoea and subclinical enteropathogen infection and quantity and attained length in 3 month intervals, at age 2 and 5 years, and used a longitudinal model to account for temporality and time-dependent confounding., Findings: Among 1469 children who completed 2 year follow-up, 35 622 stool samples were tested and yielded valid results. Diarrhoeal episodes attributed to bacteria and parasites, but not viruses, were associated with small decreases in length after 3 months and at age 2 years. Substantial decrements in length at 2 years were associated with subclinical, non-diarrhoeal, infection with Shigella (length-for-age Z score [LAZ] reduction -0·14, 95% CI -0·27 to -0·01), enteroaggregative Escherichia coli (-0·21, -0·37 to -0·05), Campylobacter (-0·17, -0·32 to -0·01), and Giardia (-0·17, -0·30 to -0·05). Norovirus, Cryptosporidium, typical enteropathogenic E coli, and Enterocytozoon bieneusi were also associated with small decrements in LAZ. Shigella and E bieneusi were associated with the largest decreases in LAZ per log increase in quantity per g of stool (-0·13 LAZ, 95% CI -0·22 to -0·03 for Shigella; -0·14, -0·26 to -0·02 for E bieneusi). Based on these models, interventions that successfully decrease exposure to Shigella, enteroaggregative E coli, Campylobacter, and Giardia could increase mean length of children by 0·12-0·37 LAZ (0·4-1·2 cm) at the MAL-ED sites., Interpretation: Subclinical infection and quantity of pathogens, particularly Shigella, enteroaggregative E coli, Campylobacter, and Giardia, had a substantial negative association with linear growth, which was sustained during the first 2 years of life, and in some cases, to 5 years. Successfully reducing exposure to certain pathogens might reduce global stunting., Funding: Bill & Melinda Gates Foundation., (Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Optimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study., Methods: We re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0-2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level pathogen-specific attributable burdens, derived stringent quantitative cutoffs to identify aetiology for individual episodes, and created aetiology prediction scores using clinical characteristics., Findings: We analysed 6625 diarrhoeal and 30 968 non-diarrhoeal surveillance stools from 1715 children. Overall, 64·9% of diarrhoea episodes (95% CI 62·6-71·2) could be attributed to an aetiology by quantitative PCR compared with 32·8% (30·8-38·7) using the original study microbiology. Viral diarrhoea (36·4% of overall incidence, 95% CI 33·6-39·5) was more common than bacterial (25·0%, 23·4-28·4) and parasitic diarrhoea (3·5%, 3·0-5·2). Ten pathogens accounted for 95·7% of attributable diarrhoea: Shigella (26·1 attributable episodes per 100 child-years, 95% CI 23·8-29·9), sapovirus (22·8, 18·9-27·5), rotavirus (20·7, 18·8-23·0), adenovirus 40/41 (19·0, 16·8-23·0), enterotoxigenic Escherichia coli (18·8, 16·5-23·8), norovirus (15·4, 13·5-20·1), astrovirus (15·0, 12·0-19·5), Campylobacter jejuni or C coli (12·1, 8·5-17·2), Cryptosporidium (5·8, 4·3-8·3), and typical enteropathogenic E coli (5·4, 2·8-9·3). 86·2% of the attributable incidence for Shigella was non-dysenteric. A prediction score for shigellosis was more accurate (sensitivity 50·4% [95% CI 46·7-54·1], specificity 84·0% [83·0-84·9]) than current guidelines, which recommend treatment only of bloody diarrhoea to cover Shigella (sensitivity 14·5% [95% CI 12·1-17·3], specificity 96·5% [96·0-97·0])., Interpretation: Quantitative molecular diagnostics improved estimates of pathogen-specific burdens of childhood diarrhoea in the community setting. Viral causes predominated, including a substantial burden of sapovirus; however, Shigella had the highest overall burden with a high incidence in the second year of life. These data could improve the management of diarrhoea in these low-resource settings., Funding: Bill & Melinda Gates Foundation., (Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Environmental Enteric Dysfunction (EED) in children from low-income countries has been linked to linear growth declines. There is a critical need to identify sensitive and early EED biomarkers., Objective: Determine whether levels of antibodies against bacterial components flagellin (flic) and lipopolysaccharide (LPS) predict poor growth., Design/methods: In a prospective birth cohort of 380 children in rural Pakistan blood and stool samples were obtained at ages 6 and 9 months. Linear mixed effects models were used to examine longitudinal associations between quartiles of anti-flic and anti-LPS antibodies and changes in LAZ, WAZ and WLZ scores. Spearman's correlations were measured between anti-flic and anti-LPS immunoglobulins with measures of systemic/enteric inflammation and intestinal regeneration., Results: Anti-LPS IgA correlated significantly with CRP, AGP and Reg1 serum at 6mo and with MPO at 9mo. In multivariate analysis at 6mo of age, higher anti-LPS IgA levels predicted greater declines in LAZ scores over subsequent 18mo (comparing highest to lowest quartile, β (SE) change in LAZ score/year = -0.313 (0.125), p-value = 0.013). Anti-flic Ig A in the two highest quartiles measured at 9mo of age had declines in LAZ of -0.269 (0.126), p = 0.033; and -0.306 (0.129), p = 0.018 respectively, during the subsequent 18mo of life, compared to those in the lowest quartile of anti-flic IgA., Conclusions and Relevance: Elevated anti-flic IgA and anti-LPS IgA antibodies at 6 and 9mo, predict declines in linear growth. Systemic and enteric inflammation correlated with anti-LPS IgA provides mechanistic considerations for potential future interventions.