Etiology of Acute Diarrheal Disease and Antimicrobial Susceptibility Pattern in Children Younger Than 5 Years Old in Nepal.

Diarrhea is a common cause of morbidity and mortality among children younger than 5 years in developing countries. Children from 3 to 60 months of age were recruited from two hospitals in Nepal- Bharatpur Hospital, Bharatpur, and Kanti Children's Hospital, Kathmandu-in 2006 to 2009. Stool specimens collected from 1,200 children with acute diarrhea (cases) and 1,200 children without diarrhea (control subjects) were examined for a broad range of enteropathogens by standard microbiology, including microscopy, enzyme immunoassay for viral pathogens (adenovirus, astrovirus, and rotavirus) and protozoa (Giardia, Cryptosporidium, and Entamoeba histolytica), as well as by using reverse transcription real-time polymerase for norovirus. Antimicrobial susceptibility testing was performed using the disk diffusion method. Overall, rotavirus (22% versus 2%), norovirus (13% versus 7%), adenovirus (3% versus 0%), Shigella (6% versus 1%), enterotoxigenic Escherichia coli (8% versus 4%), Vibrio (7% versus 0%), and Aeromonas (9% versus 3%) were identified significantly more frequently in cases than control subjects. Campylobacter, Plesiomonas, Salmonella, and diarrheagenic E. coli (enteropathogenic, enteroinvasive, enteroaggregative) were identified in similar proportions in diarrheal and non-diarrheal stools. Campylobacter was resistant to second-generation quinolone drugs (ciprofloxacin and norfloxacin), whereas Vibrio and Shigella were resistant to nalidixic acid and trimethoprim/sulfamethoxazole. This study documents the important role of rotavirus and norovirus in acute diarrhea in children younger than 5 years, followed by the bacteria Shigella, enterotoxigenic E. coli, Vibrio cholera, and Aeromonas. Data on the prevalence and epidemiology of enteropathogens identify potential pathogens for public health interventions, whereas pathogen antibiotic resistance pattern data may provide guidance on choice of therapy in clinical settings.

Weather variables as important clinical predictors of bacterial diarrhoea among international travellers.

BACKGROUND: Clinicians and travellers often have limited tools to differentiate bacterial from non-bacterial causes of travellers' diarrhoea (TD). Development of a clinical prediction rule assessing the aetiology of TD may help identify episodes of bacterial diarrhoea and limit inappropriate antibiotic use. We aimed to identify predictors of bacterial diarrhoea among clinical, demographic and weather variables, as well as to develop and cross-validate a parsimonious predictive model. METHODS: We collected de-identified clinical data from 457 international travellers with acute diarrhoea presenting to two healthcare centres in Nepal and Thailand. We used conventional microbiologic and multiplex molecular methods to identify diarrheal aetiology from stool samples. We used random forest and logistic regression to determine predictors of bacterial diarrhoea. RESULTS: We identified 195 cases of bacterial aetiology, 63 viral, 125 mixed pathogens, 6 protozoal/parasite and 68 cases without a detected pathogen. Random forest regression indicated that the strongest predictors of bacterial over viral or non-detected aetiologies were average location-specific environmental temperature and red blood cell on stool microscopy. In 5-fold cross-validation, the parsimonious model with the highest discriminative performance had an area under the receiver operator curve of 0.73 using 3 variables with calibration intercept -0.01 (standard deviation, SD 0.31) and slope 0.95 (SD 0.36). CONCLUSIONS: We identified environmental temperature, a location-specific parameter, as an important predictor of bacterial TD, among traditional patient-specific parameters predictive of aetiology. Future work includes further validation and the development of a clinical decision-support tool to inform appropriate use of antibiotics in TD.

Diversity of Rotavirus Strains in Children; Results From a Community-Based Study in Nepal.

Objective: The objectives of this study were to describe the incidence and genetic diversity of Rotavirus (RV) infection among children up to 3 years of age in a community in Nepal. Methods: We investigated community-acquired cases of asymptomatic and symptomatic RV infections in children from birth to 36 months of age in a community-based birth cohort in Bhaktapur, Nepal. Monthly surveillance and diarrheal stool samples were collected from 240 children enrolled at birth, of which 238 completed the 3 years of follow-up. Samples were screened for rotavirus by Enzyme Immuno Assay (EIA). All RV screened positives were further genotyped by reverse transcription-polymerase chain reaction for the capsid genes VP7 and VP4. Results: In total, 5,224 stool samples were collected from 238 children, followed from birth to 36 months of age. Diarrhea occurred in 92.4% (230/238) of all children in the cohort. During the 3 years study period, RV was more frequently seen in children with symptoms (7.6%) than in non-symptomatic children (0.8%). The highest RV detection rate was found in younger children between 3 and 21 months of age. Although rotavirus is known as winter diarrhea, it was detected throughout the year except in August. The highest positivity rate was observed in the months between December and March, with a peak in January. Four common G types were seen: G2 (30%), G1 (29%), G12 (19%), and G9 (16%). The most predominant genotypes seen were G2P[4] (30%), followed by G1P[8] (27.0%), G12P[6] (14.0%), G9P[8] (10%), and remaining were mixed, partial, and untyped. Conclusion: Our study confirms that rotavirus is a common cause of gastroenteritis in young children in the community. The prevalence and pathogenicity of rotavirus infection differed by age. There was substantial variability in circulating strains in the community samples compared to samples collected from hospitals. This shows the importance of including community-based surveillance systems to monitor the diversity of circulating rotavirus strains in Nepal.

Influences on catch-up growth using relative versus absolute metrics: evidence from the MAL-ED cohort study.

BACKGROUND: Poor growth in early childhood has been considered irreversible after 2-3 years of age and has been associated with morbidity and mortality over the short-term and with poor economic and cognitive outcomes over the long-term. The MAL-ED cohort study was performed in eight low-income settings with the goal of evaluating relationships between the child's environment and experience (dietary, illness, and pathogen exposure, among others) and their growth and development. The goal of this analysis is to determine whether there are differences in the factors associated with growth from 24 to 60 months using two different metrics. METHODS: Across six MAL-ED sites, 942 children had anthropometry data at 24 and 60 months, as well as information about socioeconomic status, maternal height, gut permeability (lactulose-mannitol z-score (LMZ)), dietary intake from 9 to 24 months, and micronutrient status. Anthropometric changes were in height- or weight-for-age z-score (HAZ, WAZ), their absolute difference from the growth standard median (HAD (cm), WAD (kg)), as well as recovery from stunting/underweight. Outcomes were modeled using multivariate regression. RESULTS: At 24 months, almost half of the cohort was stunted (45%) and 21% were underweight. Among those who were stunted at 24 months (n = 426), 185 (43%) were no longer stunted at 60 months. Most children increased their HAZ from 24 to 60 months (81%), whereas fewer (33%) had positive changes in their HAD. Linear regression models indicate that girls improved less than boys from 24 to 60 months (HAZ: -0.21 (95% CI -0.27, -0.15); HAD: -0.75 (-1.07, -0.43)). Greater intestinal permeability (higher LMZ) at 0-24 months was associated with lower relative and absolute changes from 24 to 60 months (HAZ: -0.10 (-0.16, -0.04); HAD: -0.47 (-0.73, -0.21)). Maternal height (per 10 cm) was positively associated with changes (HAZ: 0.09 (0.03, 0.15); HAD: 0.45 (0.15, 0.75)). Similar relationships were identified for changes in WAZ and WAD. CONCLUSIONS: The study children demonstrated improved growth from 24 to 60 months of age, but only a subset had positive changes in HAD and WAD. The same environmental factors were associated with growth from 24 to 60 months regardless of metric used (change in HAZ or HAD, or WAZ and WAD).

Historical Analysis of the Risk of Hepatitis E and Its Complications in Pregnant Women in Nepal, 1996-1998.

Hepatitis E (HE) during pregnancy can be fatal; there are no prospective risk estimates for HE and its complications during pregnancy. We followed 2,404 pregnant women for HE and pregnancy outcomes from 1996 to 1998. Subjects from Nepal were enrolled at an antenatal clinic with pregnancy of ≤ 24 weeks. Most women (65.1%) were anti-HE virus negative. There were 16 cases of HE (6.7 per 1,000); three mothers died (18.8%) having had intrauterine fetal death (IUFD). Thirteen mothers survived: five preterm and seven full-term deliveries, one IUFD. HE among seronegative women was the sole cause of maternal death and increased the risk of IUFD (relative risk [RR]: 10.6; 95% confidence interval [CI]: 4.29-26.3) and preterm delivery (RR: 17.1, 95% CI 7.56-38.5). HE vaccination of females in at-risk regions before or as they attain reproductive age would reduce their risk for preterm delivery, IUFD, and maternal death.

Genome-Wide Analysis of Differentially Expressed miRNAs and Their Associated Regulatory Networks in Lenses Deficient for the Congenital Cataract-Linked Tudor Domain Containing Protein TDRD7.

Mutations/deficiency of TDRD7, encoding a tudor domain protein involved in post-transcriptional gene expression control, causes early onset cataract in humans. While Tdrd7 is implicated in the control of key lens mRNAs, the impact of Tdrd7 deficiency on microRNAs (miRNAs) and how this contributes to transcriptome misexpression and to cataracts, is undefined. We address this critical knowledge-gap by investigating Tdrd7-targeted knockout (Tdrd7-/-) mice that exhibit fully penetrant juvenile cataracts. We performed Affymetrix miRNA 3.0 microarray analysis on Tdrd7-/- mouse lenses at postnatal day (P) 4, a stage preceding cataract formation. This analysis identifies 22 miRNAs [14 over-expressed (miR-15a, miR-19a, miR-138, miR-328, miR-339, miR-345, miR-378b, miR-384, miR-467a, miR-1224, miR-1935, miR-1946a, miR-3102, miR-3107), 8 reduced (let-7b, miR-34c, miR-298, miR-382, miR-409, miR-1198, miR-1947, miR-3092)] to be significantly misexpressed (fold-change ≥ ± 1.2, p-value < 0.05) in Tdrd7-/- lenses. To understand how these misexpressed miRNAs impact Tdrd7-/- cataract, we predicted their mRNA targets and examined their misexpression upon Tdrd7-deficiency by performing comparative transcriptomics analysis on P4 and P30 Tdrd7-/- lens. To prioritize these target mRNAs, we used various stringency filters (e.g., fold-change in Tdrd7-/- lens, iSyTE-based lens-enriched expression) and identified 98 reduced and 89 elevated mRNA targets for overexpressed and reduced miRNAs, respectively, which were classified as "top-priority" "high-priority," and "promising" candidates. For Tdrd7-/- lens overexpressed miRNAs, this approach identified 18 top-priority reduced target mRNAs: Alad, Ankrd46, Ceacam10, Dgat2, Ednrb, H2-Eb1, Klhl22, Lin7a, Loxl1, Lpin1, Npc1, Olfm1, Ppm1e, Ppp1r1a, Rgs8, Shisa4, Snx22 and Wnk2. Majority of these targets were also altered in other gene-specific perturbation mouse models (e.g., Brg1, E2f1/E2f2/E2f3, Foxe3, Hsf4, Klf4, Mafg/Mafk, Notch) of lens defects/cataract, suggesting their importance to lens biology. Gene ontology (GO) provided further insight into their relevance to lens pathology. For example, the Tdrd7-deficient lens capsule defect may be explained by reduced mRNA targets (e.g., Col4a3, Loxl1, Timp2, Timp3) associated with "basement membrane". GO analysis also identified new genes (e.g., Casz1, Rasgrp1) recently linked to lens biology/pathology. Together, these analyses define a new Tdrd7-downstream miRNA-mRNA network, in turn, uncovering several new mRNA targets and their associated pathways relevant to lens biology and offering molecular insights into the pathology of congenital cataract.

Not water, sanitation and hygiene practice, but timing of stunting is associated with recovery from stunting at 24 months: results from a multi-country birth cohort study.

OBJECTIVES: To measure the role of water, sanitation and hygiene (WASH) practices on recovery from stunting and assess the role of timing of stunting on the reversal of this phenomenon. DESIGN: Data from the MAL-ED multi-country birth cohort study was used for the current analysis. Generalised linear mixed-effects models were used to estimate the probability of reversal of stunting with WASH practice and timing of stunting as the exposures of interest. SETTING: Seven different countries across three continents. PARTICIPANTS: A total of 612 children <2 years of age. RESULTS: We found that not WASH practice but timing of stunting had statistically significant association with recovery from stunting. In comparison with the children who were stunted at 6 months, children who were stunted at 12 months had 1·9 times (ß = 0·63, P = 0·03) more chance of recovery at 24 months of age. And, children who were stunted at 18 months of age even had higher odds (adjusted OR = 3·01, ß = 1·10, P < 0·001) of recovery than children who were stunted at 6 months. Additionally, mother's height (ß = 0·59, P = 0·04) and household income (ß = 0·02, P < 0·05) showed statistically significant associations with the outcome. CONCLUSIONS: The study provided evidence for the role of timing of stunting on the recovery from the phenomenon. This novel finding indicates that the programmes to promote linear growth should be directed at the earliest possible timepoints in the course of life.

Determinants of Campylobacter infection and association with growth and enteric inflammation in children under 2 years of age in low-resource settings.

Campylobacter species infections have been associated with malnutrition and intestinal inflammation among children in low-resource settings. However, it remains unclear whether that association is specific to Campylobacter jejuni/coli. The aim of this study was to assess the association between both all Campylobacter species infections and Campylobacter jejuni/coli infections on growth and enteric inflammation in children aged 1-24 months. We analyzed data from 1715 children followed from birth until 24 months of age in the MAL-ED birth cohort study, including detection of Campylobacter species by enzyme immunoassay and Campylobacter jejuni/coli by quantitative PCR in stool samples. Myeloperoxidase (MPO) concentration in stool, used as a quantitative index of enteric inflammation, was measured. The incidence rate per 100 child-months of infections with Campylobacter jejuni/coli and Campylobacter species during 1-24 month follow up were 17.7 and 29.6 respectively. Female sex of child, shorter duration of exclusive breastfeeding, lower maternal age, mother having less than 3 living children, maternal educational level of <6 years, lack of routine treatment of drinking water, and unimproved sanitation were associated with Campylobacter jejuni/coli infection. The cumulative burden of both Campylobacter jejuni/coli infections and Campylobacter species were associated with poor growth and increased intestinal inflammation.

Enteric dysfunction and other factors associated with attained size at 5 years: MAL-ED birth cohort study findings.

BACKGROUND: Poor growth in early childhood has been associated with increased risk of mortality and morbidity, as well as long-term deficits in cognitive development and economic productivity. OBJECTIVES: Data from the MAL-ED cohort study were used to identify factors in the first 2 y of life that are associated with height-for-age, weight-for-age, and body mass index z-scores (HAZ, WAZ, BMIZ) at 5 y of age. METHODS: A total of 1017 children were followed from near birth until 5 y of age at sites in Bangladesh, Brazil, India, Nepal, Peru, South Africa, and Tanzania. Data were collected on their growth, environmental enteric dysfunction (EED), micronutrient status, enteric pathogen burden, illness prevalence, dietary intake, and various other socio-economic and environmental factors. RESULTS: EED biomarkers were related to size at 5 y. Mean lactulose:mannitol z-scores during the first 2 y of life were negatively associated with all of the growth measures (HAZ: -0.11 [95% CI: -0.19, -0.03]; WAZ: -0.16 [95% CI: -0.26, -0.06]; BMIZ: -0.11 [95% CI: -0.23, 0.0]). Myeloperoxidase was negatively associated with weight (WAZ: -0.52 [95% CI: -0.78, -0.26] and BMIZ: -0.56 [95% CI: -0.86, -0.26]); whereas α-1-antitrypsin had a negative association with HAZ (-0.28 [95% CI: -0.52, -0.04]). Transferrin receptor was positively related to HAZ (0.18 [95% CI: 0.06, 0.30]) and WAZ (0.21 [95% CI: 0.07, 0.35]). Hemoglobin was positively related to HAZ (0.06 [95% CI: 0.00, 0.12]), and ferritin was negatively related to HAZ (-0.08 [95% CI: -0.12, -0.04]). Bacterial density in stool was negatively associated with HAZ (-0.04 [95% CI: -0.08, 0.00]), but illness symptoms did not have any effect on size at 5 y. CONCLUSIONS: EED markers, bacterial density, and iron markers are associated with growth at 5 y of age. Interventions to reduce bacterial burden and EED may improve long-term growth in low-income settings.

Extended-spectrum ß-lactamase prevalence and virulence factor characterization of enterotoxigenic Escherichia coli responsible for acute diarrhea in Nepal from 2001 to 2016.

Background: Multidrug-resistant (MDR) Gram-negative bacterial species are an increasingly dangerous public health threat, and are now endemic in many areas of South Asia. However, there are a lack of comprehensive data from many countries in this region determining historic and current MDR prevalence. Enterotoxigenic Escherichia coli (ETEC) is a leading cause of both acute infant diarrhea and traveler's diarrhea in Nepal. The MDR prevalence and associated resistance mechanisms of ETEC isolates responsible for enteric infections in Nepal are largely unknown. Methods: A total of 265 ETEC isolates were obtained from acute diarrheal samples (263/265) or patient control samples (2/265) at traveler's clinics or regional hospitals in Nepal from 2001 to 2016. Isolates were screened for antibiotic resistance, to include extended spectrum beta-lactamase (ESBL) production, via the Microscan Automated Microbiology System. ETEC virulence factors, specifically enterotoxins and colonization factors (CFs), were detected using multiplex PCR, and prevalence in the total isolate population was compared to ESBL-positive isolates. ESBL-positive isolates were assessed using multiplex PCR for genetic markers potentially responsible for observed resistance. Results: A total of 118/265 (44.5%) ETEC isolates demonstrated resistance to ≥2 antibiotics. ESBL-positive phenotypes were detected in 40/265 isolates, with isolates from 2008, 2013, 2014, and 2016 demonstrating ESBL prevalence rates of 1.5, 34.5, 31.2, and 35.0% respectively. No difference was observed in overall enterotoxin characterization between the total ETEC and ESBL-positive populations. The CFs CS2 (13.6%), CS3 (25.3%), CS6 (30.2%), and CS21 (62.6%) were the most prevalent in the total ETEC population. The ESBL-positive ETEC isolates exhibited a higher association trend with the CFs CS2 (37.5%), CS3 (35%), CS6 (42.5%), and CS21 (67.5%). The primary ESBL gene identified was blaCTX-M-15 (80%), followed by blaSHV-12 (20%) and blaCTX-M-14 (2.5%). The beta-lactamase genes blaTEM-1 (40%) and blaCMY-2 (2.5%) were also identified. It was determined that 42.5% of the ESBL-positive isolates carried multiple resistance genes. Conclusion: Over 30% of ETEC isolates collected post-2013 and evaluated in this study demonstrated ESBL resistance. Persistent surveillance and characterization of enteric ETEC isolates are vital for tracking the community presence of MDR bacterial species in order to recommend effective treatment strategies and help mitigate the spread of resistant pathogens.

Nutrient intake and environmental enteric dysfunction among Nepalese children 9-24 months old-the MAL-ED birth cohort study.

BACKGROUND: Nutrient deficiencies limit the growth and turnover of intestinal mucosa, but studies assessing whether specific nutrients protect against or improve environmental enteric dysfunction (EED) are scarce. We aimed to investigate associations between nutrient intake and EED assessed by lactulose:mannitol (L:M) ratio, anti-1-antitrypsin, myeloperoxidase (MPO), and neopterin (NEO) among children 9-24 months in Bhaktapur, Nepal. METHODS: Among 231 included children, nutrient intake was assessed monthly by 24 h recalls, and 3-month usual intake was estimated using Multiple Source Method. Associations between nutrient intake and L:M ratio (measured at 15 months) were assessed using multiple linear regression, while associations between nutrient intake and fecal markers (measured quarterly) were assessed using Generalized Estimating Equations (GEE) models. RESULTS: We found that associations between nutrient intake from complementary food and L:M ratio, alpha-1-antitrypsin (AAT), MPO and NEO were generally negative but weak. The only significant associations between nutrient intake (potassium, magnesium, phosphorous, folate, and vitamin C) and markers for intestinal inflammation were found for MPO. CONCLUSION: Negative but weak associations between nutrient intake and markers of intestinal inflammation were found. Significant associations between several nutrients and MPO might merit further investigation.

Environmental Enteropathy, Micronutrient Adequacy, and Length Velocity in Nepalese Children: the MAL-ED Birth Cohort Study.

OBJECTIVES: Environmental enteropathy (EE) is likely associated with growth retardation in children, but the association between EE and length velocity z score (LVZ) has not been investigated. The objective of the study was to assess associations between fecal markers for intestinal inflammation and LVZ and whether these associations were influenced by micronutrient adequacy among 9 to 24 months old children in Bhaktapur, Nepal. METHODS: Data were divided into 5 time slots (9-12, 12-15, 15-18, 18-21, and 21-24 months). Anthropometric measurement and dietary assessment (by 24 hour recall) were performed monthly. Mean nutrient density adequacy was calculated based on nutrient density adequacy of 10 micronutrients (thiamin, riboflavin, niacin, vitamin B6, folate, vitamin C, vitamin A, calcium, iron, and zinc). Anti-1-antitrypsin (AAT), myeloperoxidase (MPO), and neopterin (NEO) were measured in stool samples collected at the beginning of each time slot. An EE score was calculated based on all 3 fecal markers. Associations between AAT, MPO, NEO and EE score and LVZ were assessed by multiple linear regression analyses and Generalized Estimating Equations models. RESULTS: Associations between fecal markers and EE score and LVZ were generally weak. EE score and MPO for 3-month and MPO for 6-month growth periods were significantly associated with LVZ from 9 to 24 months. These associations were slightly modified by mean nutrient density adequacy. CONCLUSIONS: EE score and MPO were significantly associated with LVZ in 9 to 24 months old Nepali children. Further studies to establish the usefulness of AAT, MPO, and NEO in assessing EE and growth retardation are warranted.

Tracking of infant and young child feeding practices among 9- to 24-month-old children in Nepal: the MAL-ED Birth Cohort Study.

OBJECTIVE: The present study aimed to assess infant and young child feeding (IYCF) practices and the tracking of dietary diversity score (DDS), intakes of Fe- and vitamin A-rich foods and meal frequency in a peri-urban area in Nepal. Furthermore, to explore whether sociodemographic factors were associated with tracking patterns of these IYCF practices. DESIGN: Longitudinal study. Monthly food intake was measured by 24 h recall. Four time slots were used (9-12, 13-16, 17-20 and 21-24 months). Tracking of IYCF practices was investigated using generalized estimating equations (GEE) models and Cohen's weighted kappa. Multinominal logistic regression was used to identify determinants for tracking of the IYCF practices. SETTING: Bhaktapur municipality, Nepal. SUBJECTS: Children (n 229) aged 9-24 months, randomly selected. RESULTS: Prevalence of minimum meal frequency was higher than for minimum dietary diversity at all time slots. Tracking based on absolute measures (GEE models) was moderate for DDS (0·48) and meal frequency (0·53), and low for intakes of Fe- (0·23) and vitamin A-rich (0·35) foods. Tracking based on rank measured was moderate for DDS and meal frequency, and fair for Fe- and vitamin A-rich foods. Low socio-economic status significantly increased the odds (OR; 95 % CI) of tracking of low v. high DDS (3·31; 1·44, 7·60) and meal frequency (3·46; 1·54, 7·76). CONCLUSIONS: Low tracking for intakes of Fe- and vitamin A-rich foods implies that interventions to improve these IYCF practices must address underlying causes for irregular intake to have sustainable effects.

Severely inadequate micronutrient intake among children 9-24 months in Nepal-The MAL-ED birth cohort study.

Prevalence of micronutrient deficiencies is high among infants and children in low- and middle income countries, but knowledge about nutrient adequacy across the complementary feeding period is limited. We investigated probability of adequacy (PA) of breast milk and complementary food combined and nutrient density adequacy (NDA) of complementary food and tracking of NDA over time among 229 children from 9-24 months of age in Bhaktapur, Nepal. Monthly, 24 h dietary recalls (16 in total) were performed and subgrouped into four 4-month time periods. Ten micronutrients (thiamin, riboflavin, niacin, vitamin B6 , folate, vitamin C, vitamin A, calcium, iron, and zinc) were assessed. Nutrient density was defined as the amount of a nutrient in a child's complementary food per 100 kcal, whereas NDA was the nutrient density as percentage of the context specific desired nutrient density. Tracking of NDA was investigated using generalized estimating equations models. PA for B vitamins (except riboflavin), vitamin A, calcium, iron, and zinc (low absorption group) was very low (0% to 8%) at all time slots. Median (IQR) mean PA (of all 10 micronutrients) increased from 11% (9, 15) in the second to 21% (10, 35) in the last time slot. Median value for mean nutrient density adequacy of all micronutrients varied between 42% and 52%. Finally, tracking of NDA was low (correlation <0.30) or moderate (0.30-0.60) indicating poor association between the first and subsequent measurements of NDA. These findings raise grave concerns about micronutrient adequacy among young children in Nepal. Urgent interventions are needed.

Retrospective use of next-generation sequencing reveals the presence of Enteroviruses in acute influenza-like illness respiratory samples collected in South/South-East Asia during 2010-2013.

BACKGROUND: Emerging and re-emerging respiratory pathogens represent an increasing threat to public health. Etiological determination during outbreaks generally relies on clinical information, occasionally accompanied by traditional laboratory molecular or serological testing. Often, this limited testing leads to inconclusive findings. The Armed Forces Research Institute of Medical Sciences (AFRIMS) collected 12,865 nasopharyngeal specimens from acute influenza-like illness (ILI) patients in five countries in South/South East Asia during 2010-2013. Three hundred and twenty-four samples which were found to be negative for influenza virus after screening with real-time RT-PCR and cell-based culture techniques demonstrated the potential for viral infection with evident cytopathic effect (CPE) in several cell lines. OBJECTIVE: To assess whether whole genome next-generation sequencing (WG-NGS) together with conventional molecular assays can be used to reveal the etiology of influenza negative, but CPE positive specimens. STUDY DESIGN: The supernatant of these CPE positive cell cultures were grouped in 32 pools containing 2-26 supernatants per pool. Three WG-NGS runs were performed on these supernatant pools. Sequence reads were used to identify positive pools containing viral pathogens. Individual samples in the positive pools were confirmed by qRT-PCR, RT-PCR, PCR and Sanger sequencing from the CPE culture and original clinical specimens. RESULTS: WG-NGS was an effective way to expand pathogen identification in surveillance studies. This enabled the identification of a viral agent in 71.3% (231/324) of unidentified surveillance samples, including common respiratory pathogens (100/324; 30.9%): enterovirus (16/100; 16.0%), coxsackievirus (31/100; 31.0%), echovirus (22/100; 22.0%), human rhinovirus (3/100; 3%), enterovirus genus (2/100; 2.0%), influenza A (9/100; 9.0%), influenza B, (5/100; 5.0%), human parainfluenza (4/100; 4.0%), human adenovirus (3/100; 3.0%), human coronavirus (1/100; 1.0%), human metapneumovirus (2/100; 2.0%), and mumps virus (2/100; 2.0%), in addition to the non-respiratory pathogen herpes simplex virus type 1 (HSV-1) (172/324; 53.1%) and HSV-1 co-infection with respiratory viruses (41/324; 12.7%).

Infant Nutritional Status, Feeding Practices, Enteropathogen Exposure, Socioeconomic Status, and Illness Are Associated with Gut Barrier Function As Assessed by the Lactulose Mannitol Test in the MAL-ED Birth Cohort.

The lactulose mannitol (LM) dual sugar permeability test is the most commonly used test of environmental enteropathy in developing countries. However, there is a large but conflicting literature on its association with enteric infection and host nutritional status. We conducted a longitudinal cohort using a single field protocol and comparable laboratory procedures to examine intestinal permeability in multiple, geographically diverse pediatric populations. Using a previously published systematic review to guide the selection of factors potentially associated with LM test results, we examined the relationships between these factors and mucosal breach, represented by percent lactulose excretion; absorptive area, represented by percent mannitol excretion; and gut barrier function, represented by the L/M ratio. A total of 6,602 LM tests were conducted in 1,980 children at 3, 6, 9, and 15 months old; percent lactulose excretion, percent mannitol excretion, and the L/M ratio were expressed as age- and sex-specific normalized values using the Brazil cohort as the reference population. Among the factors considered, recent severe diarrhea, lower socioeconomic status, and recent asymptomatic enteropathogen infections were associated with decreased percent mannitol excretion and higher L/M ratios. Poorer concurrent weight-for-age, infection, and recent breastfeeding were associated with increased percent lactulose excretion and increased L/M ratios. Our results support previously reported associations between the L/M ratio and factors related to child nutritional status and enteropathogen exposure. These results were remarkably consistent across sites and support the hypothesis that the frequency of these exposures in communities living in poverty leads to alterations in gut barrier function.

Use of antibiotics in children younger than two years in eight countries: a prospective cohort study.

OBJECTIVE: To describe the frequency and factors associated with antibiotic use in early childhood, and estimate the proportion of diarrhoea and respiratory illnesses episodes treated with antibiotics. METHODS: Between 2009 and 2014, we followed 2134 children from eight sites in Bangladesh, Brazil, India, Nepal, Pakistan, Peru, South Africa and the United Republic of Tanzania, enrolled in the MAL-ED birth cohort study. We documented all antibiotic use from mothers' reports at twice-weekly visits over the children's first two years of life. We estimated the incidence of antibiotic use and the associations of antibiotic use with child and household characteristics. We described treatment patterns for diarrhoea and respiratory illnesses, and identified factors associated with treatment and antibiotic class. FINDINGS: Over 1 346 388 total days of observation, 16 913 courses of antibiotics were recorded (an incidence of 4.9 courses per child per year), with the highest use in South Asia. Antibiotic treatment was given for 375/499 (75.2%) episodes of bloody diarrhoea and for 4274/9661 (44.2%) episodes of diarrhoea without bloody stools. Antibiotics were used in 2384/3943 (60.5%) episodes of fieldworker-confirmed acute lower respiratory tract illness as well as in 6608/16742 (39.5%) episodes of upper respiratory illness. Penicillins were used most frequently for respiratory illness, while antibiotic classes for diarrhoea treatment varied within and between sites. CONCLUSION: Repeated antibiotic exposure was common early in life, and treatment of non-bloody diarrhoea and non-specific respiratory illnesses was not consistent with international recommendations. Rational antibiotic use programmes may have the most impact in South Asia, where antibiotic use was highest.

Predicting Undernutrition at Age 2 Years with Early Attained Weight and Length Compared with Weight and Length Velocity.

OBJECTIVE: To estimate the abilities of weight and length velocities vs attained growth measures to predict stunting, wasting, and underweight at age 2 years. STUDY DESIGN: We analyzed data from a community-based cohort study (The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development study [MAL-ED] study) in Bhaktapur, Nepal. A total of 240 randomly selected children were enrolled at birth and followed up monthly up to age 24 months. Linear and logistic regression models were used to predict malnutrition at 2 years of age with growth velocity z scores at 0-3, 0-6, 3-6, 6-9, 6-12, and 9-12 months (using the World Health Organization Growth Standards) or attained growth at 0, 3, 6, and 12 months as predictors. RESULTS: At age 2 years, 4% of the children were wasted, 13% underweight, and 21% stunted. Children who were malnourished at age 2 years had lower mean growth z scores already at birth and throughout the study period. Anthropometric indicators in infancy were significant predictors for growth at the age of 2 years during most periods and at most ages in infancy. Weight-for-age z score, length-for-age z score, and weight-for-length z score at age 12 months had excellent areas under the curve (91-95) to predict the value of the same indicator at age 24 months. Maximum area under the curve values for weight and length velocity were somewhat lower (70-84). CONCLUSIONS: Growth measured at one time point in infancy was better correlated with undernutrition at age 2 years than growth velocity.

Dynamics and Trends in Fecal Biomarkers of Gut Function in Children from 1-24 Months in the MAL-ED Study.

Growth and development shortfalls that are disproportionately prevalent in children living in poor environmental conditions are postulated to result, at least in part, from abnormal gut function. Using data from The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) longitudinal cohort study, we examine biomarkers of gut inflammation and permeability in relation to environmental exposures and feeding practices. Trends in the concentrations of three biomarkers, myeloperoxidase (MPO), neopterin (NEO), and α-1-antitrypsin (AAT), are described from fecal samples collected during the first 2 years of each child's life. A total of 22,846 stool samples were processed during the longitudinal sampling of 2,076 children 0-24 months of age. Linear mixed models were constructed to examine the relationship between biomarker concentrations and recent food intake, symptoms of illness, concurrent enteropathogen infection, and socioeconomic status. Average concentrations of MPO, NEO, and AAT were considerably higher than published references for healthy adults. The concentration of each biomarker tended to decrease over the first 2 years of life and was highly variable between samples from each individual child. Both MPO and AAT were significantly elevated by recent breast milk intake. All three biomarkers were associated with pathogen presence, although the strength and direction varied by pathogen. The interpretation of biomarker concentrations is subject to the context of their collection. Herein, we identify that common factors (age, breast milk, and enteric infection) influence the concentration of these biomarkers. Within the context of low- and middle-income communities, we observe concentrations that indicate gut abnormalities, but more appropriate reference standards are needed.