Human milk antibodies to global pathogens reveal geographic and interindividual variations in IgA and IgG.

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).

Longevity of immune response after a single dose of typhoid conjugate vaccine against Salmonella Typhi among children in Hyderabad, Pakistan.

OBJECTIVES: Typhoid remains a persistent contributor to childhood morbidity in communities lacking sanitation infrastructure. Typhoid conjugate vaccine (TCV) is effective in reducing disease risk in vaccinees; however, the duration of protection is unknown. This study measured the longevity of immune response to TCV in children aged under 10 years in Hyderabad, Pakistan, where an outbreak of extensively drug-resistant typhoid has been ongoing. METHODS: A subset of children who received the TCV as part of the outbreak response were enrolled purposively from March 2018 to February 2019. The participants were followed up until January 2023. Blood samples were taken at baseline, 4-6 weeks, 6 months, and annually 1-4 years after vaccination to measure anti-Vi immunoglobulin (Ig) G levels using enzyme-linked immunosorbent assay. Active phone-based surveillance was performed to identify breakthrough infections. Blood culture was offered to any child with a history of fever ≥3 days within the last 7 days. A total of 81 children received a second dose of TCV in November 2019 during a catch-up campaign organized by the Sindh government. RESULTS: Nearly all participants seroconverted (802 of 837; 95.8%) at 4-6 weeks after vaccination. A total of 4 years after vaccination, 438 of 579 (75.6%) participants remained above the seroconversion threshold. The geometric mean titer (U/mL) of anti-Vi IgG at 4-6 weeks was 832.6 (95% confidence interval [CI]: 768.0-902.6); at 4 years after vaccination, the geometric mean titers in children aged 6 months to 2 years (12.6, [95% CI: 9.8-16.3]) and >2-5 years (40.1, [95% CI: 34.4-46.6]) were lower than in children aged >5-10 years (71.1, [95% CI: 59.5-85.0]). During 4 years of follow-up, nine children had culture-confirmed Salmonella Typhi infection; these infections occurred after a median duration of 3.4 years. All enteric fever cases seroconverted at 4-6 weeks after vaccination and seven (70.0%) remained seroconverted 4 years after vaccination. CONCLUSIONS: We observed 95.8% seroconversion after a single dose of TCV. There was a decay in anti-Vi IgG titers, and, at 4 years, approximately 75.6% remained seroconverted. There was a faster decay in children aged ≤2 years. Breakthrough infections were documented after a median 3.4 years after vaccination.

Machine-learning-based integrative -'omics analyses reveal immunologic and metabolic dysregulation in environmental enteric dysfunction.

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.

A shared group of bacterial taxa in the duodenal microbiota of undernourished Pakistani children with environmental enteric dysfunction.

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.

BCG activation of trained immunity is associated with induction of cross reactive COVID-19 antibodies in a BCG vaccinated population.

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.

Humoral and T cell responses to SARS-CoV-2 reveal insights into immunity during the early pandemic period in Pakistan.

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.

Clinically relevant antibiotic resistance genes are linked to a limited set of taxa within gut microbiome worldwide.

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.

Impact of diarrhoea and acute respiratory infection on environmental enteric dysfunction and growth of malnourished children in Pakistan: a longitudinal cohort study.

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.

Co-carriage of Staphylococcus aureus and Streptococcus pneumoniae among children younger than 2 years of age in a rural population in Pakistan.

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.

Essential Fatty Acid Deficiency Associates with Growth Faltering and Environmental Enteric Dysfunction in Children.

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.

Intestinal Epithelial Digestive, Transport, and Barrier Protein Expression Is Increased in Environmental Enteric Dysfunction.

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.

Quantitative Morphometry and Machine Learning Model to Explore Duodenal and Rectal Mucosal Tissue of Children with Environmental Enteric Dysfunction.

Environmental enteric dysfunction (EED) is a subclinical enteropathy prevalent in resource-limited settings, hypothesized to be a consequence of chronic exposure to environmental enteropathogens, resulting in malnutrition, growth failure, neurocognitive delays, and oral vaccine failure. This study explored the duodenal and colonic tissues of children with EED, celiac disease, and other enteropathies using quantitative mucosal morphometry, histopathologic scoring indices, and machine learning-based image analysis from archival and prospective cohorts of children from Pakistan and the United States. We observed villus blunting as being more prominent in celiac disease than in EED, as shorter lengths of villi were observed in patients with celiac disease from Pakistan than in those from the United States, with median (interquartile range) lengths of 81 (73, 127) µm and 209 (188, 266) µm, respectively. Additionally, per the Marsh scoring method, celiac disease histologic severity was increased in the cohorts from Pakistan. Goblet cell depletion and increased intraepithelial lymphocytes were features of EED and celiac disease. Interestingly, the rectal tissue from cases with EED showed increased mononuclear inflammatory cells and intraepithelial lymphocytes in the crypts compared with controls. Increased neutrophils in the rectal crypt epithelium were also significantly associated with increased EED histologic severity scores in duodenal tissue. We observed an overlap between diseased and healthy duodenal tissue upon leveraging machine learning image analysis. We conclude that EED comprises a spectrum of inflammation in the duodenum, as previously described, and the rectal mucosa, warranting the examination of both anatomic regions in our efforts to understand and manage EED.

Differential coverage for vaccines in the expanded program on immunization (EPI) among children in rural Pakistan.

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.

Environmental enteric dysfunction: gut and microbiota adaptation in pregnancy and infancy.

Environmental enteric dysfunction (EED) is a subclinical syndrome of intestinal inflammation, malabsorption and barrier disruption that is highly prevalent in low- and middle-income countries in which poverty, food insecurity and frequent exposure to enteric pathogens impair growth, immunity and neurodevelopment in children. In this Review, we discuss advances in our understanding of EED, intestinal adaptation and the gut microbiome over the 'first 1,000 days' of life, spanning pregnancy and early childhood. Data on maternal EED are emerging, and they mirror earlier findings of increased risks for preterm birth and fetal growth restriction in mothers with either active inflammatory bowel disease or coeliac disease. The intense metabolic demands of pregnancy and lactation drive gut adaptation, including dramatic changes in the composition, function and mother-to-child transmission of the gut microbiota. We urgently need to elucidate the mechanisms by which EED undermines these critical processes so that we can improve global strategies to prevent and reverse intergenerational cycles of undernutrition.

SARS-CoV-2 spike conformation determines plasma neutralizing activity elicited by a wide panel of human vaccines.

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 S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 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.

Streptococcus mutans carriage in the saliva of mothers and its association with dental caries and Streptococcus mutans carriage in the saliva of children between 6 and 30 months old in a low-income setting in Karachi, Pakistan.

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.

Frequency of bystander exposure to antibiotics for enteropathogenic bacteria among young children in low-resource settings.

Children in low-resource settings carry enteric pathogens asymptomatically and are frequently treated with antibiotics, resulting in opportunities for pathogens to be exposed to antibiotics when not the target of treatment (i.e., bystander exposure). We quantified the frequency of bystander antibiotic exposures for enteric pathogens and estimated associations with resistance among children in eight low-resource settings. We analyzed 15,697 antibiotic courses from 1,715 children aged 0 to 2 y from the MAL-ED birth cohort. We calculated the incidence of bystander exposures and attributed exposures to respiratory and diarrheal illnesses. We associated bystander exposure with phenotypic susceptibility of E. coli isolates in the 30 d following exposure and at the level of the study site. There were 744.1 subclinical pathogen exposures to antibiotics per 100 child-years. Enteroaggregative Escherichia coli was the most frequently exposed pathogen, with 229.6 exposures per 100 child-years. Almost all antibiotic exposures for Campylobacter (98.8%), enterotoxigenic E. coli (95.6%), and typical enteropathogenic E. coli (99.4%), and the majority for Shigella (77.6%), occurred when the pathogens were not the target of treatment. Respiratory infections accounted for half (49.9%) and diarrheal illnesses accounted for one-fourth (24.6%) of subclinical enteric bacteria exposures to antibiotics. Bystander exposure of E. coli to class-specific antibiotics was associated with the prevalence of phenotypic resistance at the community level. Antimicrobial stewardship and illness-prevention interventions among children in low-resource settings would have a large ancillary benefit of reducing bystander selection that may contribute to antimicrobial resistance.

Performance of Machine Learning Classifiers in Classifying Stunting among Under-Five Children in Zambia.

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.

Burden, Clinical Characteristics, Risk Factors, and Seasonality of Adenovirus 40/41 Diarrhea in Children in Eight Low-Resource Settings.

Background: The application of molecular diagnostics has identified enteric group adenovirus serotypes 40 and 41 as important causes of diarrhea in children. However, many aspects of the epidemiology of adenovirus 40/41 diarrhea have not been described. Methods: We used data from the 8-site Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project birth cohort study to describe site- and age-specific incidence, risk factors, clinical characteristics, and seasonality. Results: The incidence of adenovirus 40/41 diarrhea was substantially higher by quantitative polymerase chain reaction than enzyme immunoassay and peaked at ∼30 episodes per 100 child-years in children aged 7-15 months, with substantial variation in incidence between sites. A significant burden was also seen in children 0-6 months of age, higher than other viral etiologies with the exception of rotavirus. Children with adenovirus 40/41 diarrhea were more likely to have a fever than children with norovirus, sapovirus, and astrovirus (adjusted odds ratio [aOR], 1.62; 95% CI, 1.16-2.26) but less likely than children with rotavirus (aOR, 0.66; 95% CI, 0.49-0.91). Exclusive breastfeeding was strongly protective against adenovirus 40/41 diarrhea (hazard ratio, 0.64; 95% CI, 0.48-0.85), but no other risk factors were identified. The seasonality of adenovirus 40/41 diarrhea varied substantially between sites and did not have clear associations with seasonal variations in temperature or rainfall. Conclusions: This study supports the situation of adenovirus 40/41 as a pathogen of substantial importance, especially in infants. Fever was a distinguishing characteristic in comparison to other nonrotavirus viral etiologies, and promotion of exclusive breastfeeding may reduce the high observed burden in the first 6 months of life.