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.

Exploring the path to polio eradication: insights from consecutive seroprevalence surveys among Pakistani children.

Introduction: After trivalent oral poliovirus vaccine (tOPV) cessation, Pakistan has maintained immunity to type 2 poliovirus by administering inactivated polio vaccine (IPV) in routine immunization, alongside monovalent OPV type 2 (mOPV2) and IPV in supplementary immunization activities (SIAs). This study assesses the change in poliovirus type 2 immunity after tOPV withdrawal and due to SIAs with mOPV2 and IPV among children aged 6-11 months. Methods: Three cross-sectional sequential serological surveys were conducted in 12 polio high-risk areas of Pakistan. 25 clusters from each geographical stratum were selected utilizing probability proportional to size. Results: Seroprevalence of type 2 poliovirus was 49%, with significant variation observed among surveyed areas; <30% in Pishin, >80% in Killa Abdullah, Mardan & Swabi, and Rawalpindi. SIAs with IPV improved immunity from 38 to 57% in Karachi and 60 to 88% in Khyber. SIAs with IPV following mOPV2 improved immunity from 62 to 65% in Killa Abdullah, and combined mOPV2 and IPV SIAs in Pishin improved immunity from 28 to 89%. Results also reflected that immunity rates for serotypes 1 and 3 were consistently above 90% during all three phases and across all geographical areas. Conclusion: The study findings highlight the importance of implementing effective vaccination strategies to prevent the re-emergence of poliovirus. Moreover, the results provide crucial information for policymakers working toward achieving global polio eradication.

Microbiological Methods Used in the Enterics for Global Health Shigella Surveillance Study.

Background: Shigella is a major cause of diarrhea in young children worldwide. Multiple vaccines targeting Shigella are in development, and phase 3 clinical trials are imminent to determine efficacy against shigellosis. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study is designed to determine the incidence of medically attended shigellosis in 6- to 35-month-old children in 7 resource-limited settings. Here, we describe the microbiological methods used to isolate and identify Shigella. We developed a standardized laboratory protocol for isolation and identification of Shigella by culture. This protocol was implemented across all 7 sites, ensuring consistency and comparability of results. Secondary objectives of the study are to determine the antibiotic resistance profiles of Shigella, compare isolation of Shigella from rectal swabs versus whole stool, and compare isolation of Shigella following transport of rectal swabs in Cary-Blair versus a modified buffered glycerol saline transport medium. Conclusions: Data generated from EFGH using culture methods described herein can potentially be used for microbiological endpoints in future phase 3 clinical trials to evaluate vaccines against shigellosis and for other clinical and public health studies focused on these organisms.

Evaluation of Fecal Inflammatory Biomarkers to Identify Bacterial Diarrhea Episodes: Systematic Review and Protocol for the Enterics for Global Health Shigella Surveillance Study.

Background: The measurement of fecal inflammatory biomarkers among individuals presenting to care with diarrhea could improve the identification of bacterial diarrheal episodes that would benefit from antibiotic therapy. We reviewed prior literature in this area and describe our proposed methods to evaluate 4 biomarkers in the Enterics for Global Health (EFGH) Shigella surveillance study. Methods: We systematically reviewed studies since 1970 from PubMed and Embase that assessed the diagnostic characteristics of inflammatory biomarkers to identify bacterial diarrhea episodes. We extracted sensitivity and specificity and summarized the evidence by biomarker and diarrhea etiology. In EFGH, we propose using commercial enzyme-linked immunosorbent assays to test for myeloperoxidase, calprotectin, lipocalin-2, and hemoglobin in stored whole stool samples collected within 24 hours of enrollment from participants in the Bangladesh, Kenya, Malawi, Pakistan, Peru, and The Gambia sites. We will develop clinical prediction scores that incorporate the inflammatory biomarkers and evaluate their ability to identify Shigella and other bacterial etiologies of diarrhea as determined by quantitative polymerase chain reaction (qPCR). Results: Forty-nine studies that assessed fecal leukocytes (n = 39), red blood cells (n = 26), lactoferrin (n = 13), calprotectin (n = 8), and myeloperoxidase (n = 1) were included in the systematic review. Sensitivities were high for identifying Shigella, moderate for identifying any bacteria, and comparable across biomarkers. Specificities varied depending on the outcomes assessed. Prior studies were generally small, identified red and white blood cells by microscopy, and used insensitive gold standard diagnostics, such as conventional bacteriological culture for pathogen detection. Conclusions: Our evaluation of inflammatory biomarkers to distinguish diarrhea etiologies as determined by qPCR will provide an important addition to the prior literature, which was likely biased by the limited sensitivity of the gold standard diagnostics used. We will determine whether point-of-care biomarker tests could be a viable strategy to inform treatment decision making and increase appropriate targeting of antibiotic treatment to bacterial diarrhea episodes.

Safety and efficacy of sequential treatments for postmenopausal osteoporosis: a network meta-analysis of randomised controlled trials.

Background: The sequential anti-osteoporotic treatment for women with postmenopausal osteoporosis (PMO) is important, but the order in which different types of drugs are used is confusing and controversial. Therefore, we performed a network meta-analysis to compare the efficacy and safety of available sequential treatments to explore the most efficacious strategy for long-term management of osteoporosis. Methods: In this network meta-analysis, we searched the PubMed, EMBASE, Web of Science, the Cochrane Library, and ClinicalTrials.gov from inception to September 19, 2023 to identify randomised controlled trials comparing sequential treatments for women with PMO. The identified trials were screened by reading the title and abstract, and only randomised clinical trials involving sequential anti-osteoporotic treatments and reported relevant outcomes for PMO were included. The main outcomes included vertebral fracture risk, the percentage change in bone mineral density (BMD) in different body parts, and all safety indicators in the stage after switching treatment. A frequentist network meta-analysis was performed using the multivariate random effects method and evaluated using the surface under the cumulative ranking curve (SUCRA). Certainty of evidence was assessed using the Confidence in the Network Meta-Analysis (CINeMA) framework. This study is registered with PROSPERO: CRD42022360236. Findings: A total of 19 trials comprising 18,416 participants were included in the study. Five different sequential treatments were investigated as the main interventions and compared to the corresponding control groups. The intervention groups in this study comprised the following treatment switch protocols: switching from an anabolic agent (AB) to an anti-resorptive agent (AR) (ABtAR), transitioning from one AR to another AR (ARtAAR), shifting from an AR to an AB (ARtAB), switching from an AB to a combined treatment of AB and AR (ABtC), and transitioning from an AR to a combined treatment (ARtC). A significant reduction in the incidence of vertebral fractures was observed in ARtC, ABtAR and ARtAB in the second stage, and ARtC had the lowest incidence with 81.5% SUCRA. ARtAAR and ABtAR were two effective strategies for preventing fractures and improving BMD in other body parts. Especially, ARtAAR could improve total hip BMD with the highest 96.1% SUCRA, and ABtAR could decrease the risk of total fractures with the highest 94.3% SUCRA. Almost no difference was observed in safety outcomes in other comparisons. Interpretation: Our findings suggested that the ARtAAR and ABtAR strategy are the effective and safe sequential treatment for preventing fracture and improving BMD for PMO. ARtC is more effective in preventing vertebral fractures. Funding: The National Natural Science Foundation of China (82170900, 81970762), the Hunan Administration of Traditional Chinese Medicine, and the Hunan Province High-level Health Talents "225" Project.

Effect of Maternal and Newborn Care Service Package on Perinatal and Newborn Mortality: A Cluster Randomized Clinical Trial.

Importance: In resource-constrained settings where the neonatal mortality rate (NMR) is high due to preventable causes and health systems are underused, community-based interventions can increase newborn survival by improving health care practices. Objectives: To develop and evaluate the effectiveness of a community-based maternal and newborn care services package to reduce perinatal and neonatal mortality in rural Pakistan. Design, Setting, and Participants: This cluster randomized clinical trial was conducted between November 1, 2012, and December 31, 2013, in district Rahim Yar Khan in the province of Punjab. A cluster was defined as an administrative union council. Any consenting pregnant resident of the study area, regardless of gestational age, was enrolled. An ongoing pregnancy surveillance system identified 12 529 and 12 333 pregnancies in the intervention and control clusters, respectively; 9410 pregnancies were excluded from analysis due to continuation of pregnancy at the end of the study, loss to follow-up, or miscarriage. Participants were followed up until the 40th postpartum day. Statistical analysis was performed from January to May 2014. Intervention: A maternal and newborn health pack, training for community- and facility-based health care professionals, and community mobilization through counseling and education sessions. Main Outcomes and Measures: The primary outcome was perinatal mortality, defined as stillbirths per 1000 births and neonatal death within 7 days per 1000 live births. The secondary outcome was neonatal mortality, defined as death within 28 days of life per 1000 live births. Systematic random sampling was used to allocate 10 clusters each to intervention and control groups. Analysis was conducted on a modified intention-to-treat basis. Results: For the control group vs the intervention group, the total number of households was 33 188 vs 34 315, the median number of households per cluster was 3092 (IQR, 3018-3467) vs 3469 (IQR, 3019-4075), the total population was 229 155 vs 234 674, the mean (SD) number of residents per household was 6.9 (9.5) vs 6.8 (9.6), the number of males per 100 females (ie, the sex ratio) was 104.2 vs 103.7, and the mean (SD) number of children younger than 5 years per household was 1.0 (4.2) vs 1.0 (4.3). Altogether, 7598 births from conrol clusters and 8017 births from intervention clusters were analyzed. There was no significant difference in perinatal mortality between the intervention and control clusters (rate ratio, 0.86; 95% CI, 0.69-1.08; P = .19). The NMR was lower among the intervention than the control clusters (39.2/1000 live births vs 52.2/1000 live births; rate ratio, 0.75; 95% CI, 0.58-0.95; P = .02). The frequencies of antenatal visits and facility births were similar between the 2 groups. However, clean delivery practices were higher among intervention clusters than control clusters (63.2% [2284 of 3616] vs 13.2% [455 of 3458]; P < .001). Chlorhexidine use was also more common among intervention clusters than control clusters (55.9% [4271 of 7642] vs 0.3% [19 of 7203]; P < .001). Conclusions and Relevance: This pragmatic cluster randomized clinical trial demonstrated a reduction in NMR that occurred in the background of improved household intrapartum and newborn care practices. However, the effect of the intervention on antenatal visits, facility births, and perinatal mortality rates was inconclusive, highlighting areas requiring further research. Nevertheless, the improvement in NMR underscores the effectiveness of community-based programs in low-resource settings. Trial Registration: ClinicalTrials.gov Identifier: NCT01751945.

Risk of incident chronic kidney disease in metabolically healthy obesity and metabolically unhealthy normal weight: A systematic review and meta-analysis.

Studies have reported inconsistent results about the risk of incident chronic kidney disease (CKD) in people with metabolically healthy obesity (MHO). We designed this systematic review and meta-analysis to evaluate the risk of developing CKD in people with MHO and metabolically unhealthy normal weight (MUNW). We used a predefined search strategy to retrieve eligible studies from multiple databases up to June 20, 2022. Random-effects model meta-analyses were implied to estimate the overall hazard ratio (HR) of incident CKD in obesity phenotypes. Eight prospective cohort studies, including approximately 5 million participants with a median follow-up ranging between 3 and 14 years, were included in this meta-analysis. Compared to the metabolically healthy normal weight (MHNW), the mean differences in cardiometabolic and renal risk factors in MHO, MUNW, and metabolically unhealthy obesity (MUO) were evaluated with overall HR of 1.42, 1.49, and 1.84, respectively. Compared to MHNW, the mean estimated glomerular filtration rate (eGFR) and high-density lipoprotein (HDL) were significantly lower, and low-density lipoprotein (LDL), blood pressure, blood glucose, and triglycerides were higher in MHO and MUNW. In conclusion, MHO and MUNW are not benign conditions and pose a higher risk for incident CKD. Obesity, whether in the presence or absence of metabolic health, is a risk factor for CKD.

Exploring the long-term seroprevalence of SARS-CoV-2 antibodies in infants born to women with clinical or laboratory-confirmed COVID-19.

INTRODUCTION: Infants are at a higher risk of severe illness with COVID-19 infection compared to older children. While COVID-19 vaccination is not recommended for young infants, they can acquire maternally-derived anti-SARS-CoV-2 antibodies passively through the placenta and breastmilk. We described the persistence of infection-induced maternal antibodies in infant circulation at 9-12 months of age. METHODOLOGY: This was a cross-sectional study nested within the INTERCOVID multinational cohort study. For each COVID positive pregnant woman, two unmatched consecutive COVID negative pregnant women were enrolled between April and September 2020. Women with a positive PCR test, radiographic signs consistent with COVID-19, or at least 2 predefined symptoms of COVID-19 were considered as COVID positive. For this nested cross-sectional study, all COVID positive and either one of the COVID negative participants recruited from the Aga Khan University, Pakistan were approached 9-12 months after delivery, and maternal and infant sera were collected for antibody detection. RESULTS: Altogether, 83 mothers provided consent, of whom 32 (38.6 %) were COVID positive and 51 (61.4 %) were COVID negative during pregnancy. Anti-SARS-CoV-2 antibodies were present in 13 (41 %) infants born to COVID positive and 19 (39 %) infants born to COVID negative mothers (p = 0.87). The presence of reactive antibodies in infants at follow-up was associated with maternal antibodies at follow-up (OR:9.50, 95 % CI:2.03-44.42; p = 0.004). COVID infection occurred in 3 (6 %) infants born to COVID negative mothers while no infant born to a COVID positive mother had a history of infection (p = 0.27). CONCLUSION: The presence of reactive anti-SARS-CoV-2 antibodies in infants at 9-12 months of age is associated with maternal seropositivity 9-12 months after delivery rather than maternal infection during pregnancy. Further studies are required to validate these findings and assess whether passive immunity in infants is protective against COVID-19 infection.

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.

An outbreak of Salmonella Typhimurium associated with the consumption of raw liver at an Eid al-Adha celebration in Wales (UK), July 2021.

In July 2021, Public Health Wales received two notifications of salmonella gastroenteritis. Both cases has attended the same barbecue to celebrate Eid al-Adha, two days earlier. Additional cases attending the same barbecue were found and an outbreak investigation was initiated. The barbecue was attended by a North African community's social network. On same day, smaller lunches were held in three homes in the social network. Many people attended both a lunch and the barbecue. Cases were defined as someone with an epidemiological link to the barbecue and/or lunches with diarrhoea and/or vomiting with date of onset following these events. We undertook a cohort study of 36 people attending the barbecue and/or lunch, and a nested case-control study using Firth logistic regression. A communication campaign, sensitive towards different cultural practices, was developed in collaboration with the affected community. Consumption of a traditional raw liver dish, 'marrara', at the barbecue was the likely vehicle for infection (Firth logistic regression, aOR: 49.99, 95%CI 1.71-1461.54, p = 0.02). Meat and offal came from two local butchers (same supplier) and samples yielded identical whole genome sequences as cases. Future outbreak investigations should be relevant to the community affected by considering dishes beyond those found in routine questionnaires.

Rare but critical: Aberrant vascular communication leading to multiorgan ischemia after prophylactic gastroduodenal artery embolization for refractory upper gastrointestinal bleeding.

Upper gastrointestinal bleeding (UGIB) carries a high risk of morbidity and mortality despite recent improvements in diagnosis and management. Many patients failed to respond to initial endoscopic and medical management. Transcatheter arterial embolization (TAE) is now considered the next line of therapy over surgery in refractory UGIB because of its good safety profile and high technical and clinical success rate. We discuss the case of a 66-year-old female patient who presented with massive UGIB and had TAE after endoscopic hemostasis failed. She developed widespread ischemia involving multiple organs following the procedure, including the liver, gallbladder, pancreas, spleen, and small intestine, as a result of an abnormal communication between the gastroduodenal artery (GDA) and superior mesenteric artery (SMA), resulting in PVA particle reflux and widespread ischemic injury. It is important to carefully evaluate the vascular anatomic variations before the procedure to avoid potential complications of ischemia.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Salmonella Typhi (Typhi) is a type of bacteria that causes typhoid fever. More than 110,000 people die from this disease each year, predominantly in areas of sub-Saharan Africa and South Asia with limited access to safe water and sanitation. Clinicians use antibiotics to treat typhoid fever, but scientists worry that the spread of antimicrobial-resistant Typhi could render the drugs ineffective, leading to increased typhoid fever mortality. The World Health Organization has prequalified two vaccines that are highly effective in preventing typhoid fever and may also help limit the emergence and spread of resistant Typhi. In low resource settings, public health officials must make difficult trade-off decisions about which new vaccines to introduce into already crowded immunization schedules. Understanding the local burden of antimicrobial-resistant Typhi and how it is spreading could help inform their actions. The Global Typhoid Genomics Consortium analyzed 13,000 Typhi genomes from 110 countries to provide a global overview of genetic diversity and antimicrobial-resistant patterns. The analysis showed great genetic diversity of the different strains between countries and regions. For example, the H58 Typhi variant, which is often drug-resistant, has spread rapidly through Asia and Eastern and Southern Africa, but is less common in other regions. However, distinct strains of other drug-resistant Typhi have emerged in other parts of the world. Resistance to the antibiotic ciprofloxacin was widespread and accounted for over 85% of cases in South Africa. Around 70% of Typhi from Pakistan were extensively drug-resistant in 2020, but these hard-to-treat variants have not yet become established elsewhere. Variants that are resistant to both ciprofloxacin and ceftriaxone have been identified, and azithromycin resistance has also appeared in several different variants across South Asia. The Consortium's analyses provide valuable insights into the global distribution and transmission patterns of drug-resistant Typhi. Limited genetic data were available fromseveral regions, but data from travel-associated cases helped fill some regional gaps. These findings may help serve as a starting point for collective sharing and analyses of genetic data to inform local public health action. Funders need to provide ongoing supportto help fill global surveillance data gaps.

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.

Determinants of Stunting among Children under Five in Pakistan.

INTRODUCTION: Child stunting remains a public health concern. It is characterized as poor cognitive and physical development in children due to inadequate nutrition during the first 1000 days of life. Across south Asia, Pakistan has the second-highest prevalence of stunting. This study assessed the most recent nationally representative data, the National Nutrition Survey (NNS) 2018, to identify the stunting prevalence and determinants among Pakistani children under five. METHODS: The NNS 2018, a cross-sectional household-level survey, was used to conduct a secondary analysis. Data on malnutrition, dietary practices, and food insecurity were used to identify the prevalence of stunting among children under five years in terms of demographic, socioeconomic, and geographic characteristics. The prevalence of stunting was calculated using the World Health Organization (WHO) height for age z-score references. Univariate and multivariable logistic regressions were conducted to identify the factors associated with child stunting. RESULTS: The analysis showed that out of 52,602 children under five, 40.0% were found to be stunted. Male children living in rural areas were more susceptible to stunting. Furthermore, stunting was more prevalent among children whose mothers had no education, were between 20 and 34, and were employed. In the multivariable logistic regression, male children (AOR = 1.08, 95% CI [1.04-1.14], p < 0.001) from rural areas (AOR = 1.07, 95% CI [1.01-1.14], p = 0.014), with the presence of diarrhea in the last two weeks (AOR = 1.15, 95% CI [1.06-1.25], p < 0.001) and mothers who had no education (AOR = 1.57, 95% CI [1.42-1.73], p < 0.001) or lower levels of education (primary: AOR = 1.35, 95% CI [1.21-1.51], p < 0.001; middle: AOR = 1.29, 95% CI [1.15-1.45], p < 0.001), had higher odds of stunting. Younger children aged < 6 months (AOR = 0.53, 95% CI [0.48-0.58], p < 0.001) and 6-23 months (AOR = 0.89, 95% CI [0.84-0.94], p < 0.001), with mothers aged 35-49 years (AOR = 0.78, 95% CI [0.66-0.92], p = 0.003), had lower odds of stunting. At the household level, the odds of child stunting were higher in lower-income households (AOR = 1.64, 95% CI [1.46-1.83], p < 0.001) with ≥ 7 members (AOR = 1.09, 95% CI [1.04-1.15], p < 0.001), with no access to improved sanitation facilities (AOR = 1.14, 95% CI [1.06-1.22], p < 0.001) and experiencing severe food insecurity (AOR = 1.07, 95% CI [1.01-1.14], p = 0.02). CONCLUSION: Child stunting in Pakistan is strongly associated with various factors, including gender, age, diarrhea, residence, maternal age and education, household size, food and wealth status, and access to sanitation. To address this, interventions must be introduced to make locally available food and nutritious supplements more affordable, improve access to safe water and sanitation, and promote female education for long-term reductions in stunting rates.

Hereditary severe insulin resistance syndrome: Pathogenesis, pathophysiology, and clinical management.

Severe insulin resistance has been linked to some of the most globally prevalent disorders, such as diabetes mellitus, nonalcoholic fatty liver disease, polycystic ovarian syndrome, and hypertension. Hereditary severe insulin resistance syndrome (H-SIRS) is a rare disorder classified into four principal categories: primary insulin receptor defects, lipodystrophies, complex syndromes, and obesity-related H-SIRS. Genes such as INSR, AKT2, TBC1D4, AGPAT2, BSCL2, CAV1, PTRF, LMNA, PPARG, PLIN1, CIDEC, LIPE, PCYT1A, MC4R, LEP, POMC, SH2B1, RECQL2, RECQL3, ALMS1, PCNT, ZMPSTE24, PIK3R1, and POLD1 have been linked to H-SIRS. Its clinical features include insulin resistance, hyperglycemia, hyperandrogenism, severe dyslipidemia, fatty liver, abnormal topography of adipose tissue, and low serum leptin and adiponectin levels. Diagnosis of H-SIRS is based on the presence of typical clinical features associated with the various H-SIRS forms and the identification of mutations in H-SIRS-linked genes by genetic testing. Diet therapy, insulin sensitization, exogenous insulin therapy, and leptin replacement therapy have widely been adopted to manage H-SIRS. The rarity of H-SIRS, its highly variable clinical presentation, refusal to be tested for genetic mutations by patients' family members who are not severely sick, unavailability of genetic testing, and testing expenses contribute to the delayed or underdiagnoses of H-SIRS. Early diagnosis facilitates early management of the condition, which results in improved glycemic control and delayed onset of diabetes and other complications related to severe insulin resistance. The use of updated genetic sequencing technologies is recommended, and long-term studies are required for genotype-phenotype differentiation and formulation of diagnostic and treatment protocols.

Fat distribution measurements by chemical shift-encoded transition region extraction predict the risk of hyperglycaemia, dyslipidaemia and metabolic syndrome in mice.

Metabolically healthy or unhealthy obesity is closely related to metabolic syndrome (MetS). To validate a more accurate diagnostic method for obesity that reflects the risk of metabolic disorders in a pre-clinical mouse model, C57BL/6J mice were fed high-sucrose-high-fat and chow diets for 12 weeks to induce obesity. MRI was performed and analysed by chemical shift-encoded fat-water separation based on the transition region extraction method. Abdominal fat was divided into upper and lower abdominal regions at the horizontal lower border of the liver. Blood samples were collected, and the glucose level, lipid profile, liver function, HbA1c and insulin were tested. k-means clustering and stepwise logistic regression were applied to validate the diagnosis of hyperglycaemia, dyslipidaemia and MetS, and to ascertain the predictive effect of MRI-derived parameters to the metabolic disorders. Pearson or Spearman correlation was used to assess the relationship between MRI-derived parameters and metabolic traits. The receiver-operating characteristic curve was used to evaluate the diagnostic effect of each logistic regression model. A two-sided p value less than 0.05 was considered to indicate statistical significance for all tests. We made the precise diagnosis of obesity, dyslipidaemia, hyperglycaemia and MetS in mice. In all, 14 mice could be diagnosed as having MetS, and the levels of body weight, HbA1c, triglyceride, total cholesterol and low-density lipoprotein cholesterol were significantly higher than in the normal group. Upper abdominal fat better predicted dyslipidaemia (odds ratio, OR = 2.673; area under the receiver-operating characteristic curve, AUCROC = 0.9153) and hyperglycaemia (OR = 2.456; AUCROC = 0.9454), and the abdominal visceral adipose tissue (VAT) was better for predicting MetS risk (OR = 1.187; AUCROC = 0.9619). We identified the predictive effect of fat volume and distribution in dyslipidaemia, hyperglycaemia and MetS. The upper abdominal fat played a better predictive role for the risk of dyslipidaemia and hyperglycaemia, and the abdominal VAT played a better predictive role for the risk of MetS.

Notch1 is a marker for in situ resting osteocytes in a 3-dimensional gel culture model.

PURPOSE: Osteocytes in vivo exhibit different functional states, but no specific marker to distinguish these is currently available. MATERIALS AND METHODS: To simulate the differentiation process of pre-osteoblasts to osteocytes in vitro, MC3T3-E1 cells were cultured on type I collagen gel and a three-dimensional (3D) culture system was established. The Notch expression of osteocyte-like cells in 3D culture system was compared with that of in situ osteocytes in bone tissues. RESULTS: Immunohistochemistry demonstrated that Notch1 was not detected in "resting" in situ osteocytes, but was detected in normal cultured osteocyte-like cell line MLO-Y4. Osteocytes obtained from conventional osteogenic-induced osteoblasts and long-term cultured MLO-Y4 cells could not replicate the Notch1 expression pattern from in situ osteocytes. From day 14-35 of osteogenic induction, osteoblasts in 3D culture system gradually migrated into the gel to form canaliculus-like structures similar to bone canaliculus. On day 35, stellate-shaped osteocyte-like cells were observed, and expression of DMP1 and SOST, but not Runx2, was detected. Notch1 was not detected by immunohistochemistry, and Notch1 mRNA level was not significantly different from that of in situ osteocytes. In MC3T3-E1 cells, down-regulation of Notch2 increased Notch1, Notch downstream genes (ß-catenin and Nfatc1), and Dmp1. In MLO-Y4 cells, Notch2 decreased after Notch1 siRNA transfection. Downregulation of Notch1 or Notch2 decreased Nfatc1, ß-catenin, and Dmp1, and increased Sost. CONCLUSIONS: We established "resting state" osteocytes using an in vitro 3D model. Notch1 can be a useful marker to help differentiate the functional states of osteocytes (activated vs. resting state).

Plasticity of adipose tissues in response to fasting and refeeding declines with aging in mice.

To explore the plasticity of adipose tissues, C57BL/6J mice at the age of 1 month, 3 months, and 15 months corresponding to adolescence, adulthood, and middle-aged transitional period, respectively, were fasted and refed subsequently at different times. Body adipose tissues ratio (BATR) was calculated, the morphology of adipose tissue and the area of adipocytes were observed by histological analysis, and the mitochondria in adipocytes were observed under the transmission electron microscope. Furthermore, the expression levels of Ucp-1, Cidea, Cox7a1, Cpt-1m, Atgl, and Hsl were detected by qRT-PCR. Our results showed a significant increase in the adipocytes area and body visceral adipose tissue (VAT) ratio in all groups of mice with aging. Moreover, body mesenteric white adipose tissue (mWAT) ratio decreased the most after 72 h fasting. In the middle-aged transitional mice, the white adipocytes did not decrease until 72 h fasting, and most of them still appeared as unaffected unilocular cells. Besides, the number of mitochondria and the expression of Ucp-1, Cidea, Cox7a1, Cpt-1m, Atgl and Hsl were lower in these mice. After 72h refeeding, the body subcutaneous white adipose tissue (sWAT) ratio returned to normal, while the VAT kept decreasing. The above results indicated an impairment in adipose tissue plasticity in mice with aging, suggesting that age modulated the metabolic adaptiveness of adipose tissues in mice.