Childhood acute illness and nutrition (CHAIN) network: A protocol for a multi-site prospective cohort study to identify modifiable risk factors for mortality among acutely ill children in Africa and Asia

Introduction: Children admitted to hospitals in resource-poor settings remain at risk of both inpatient and post-discharge mortality. While known risk factors such as young age and nutritional status can identify children at risk, they do not provide clear mechanistic targets for intervention. The Childhood Acute Illness and Nutrition (CHAIN) cohort study aims to characterise the biomedical and social risk factors for mortality in acutely ill children in hospitals and after discharge to identify targeted interventions to reduce mortality.Methods and analysis: The CHAIN network is currently undertaking a multi-site, prospective, observational cohort study, enrolling children aged 1 week to 2 years at admission to hospitals at nine sites located in four African and two South Asian countries. The CHAIN Network supports the sites to provide care according to national and international guidelines. Enrolment is stratified by anthropometric status and children are followed throughout hospitalisation and for 6 months after discharge. Detailed clinical, demographic, anthropometric, laboratory and social exposures are assessed. Scheduled visits are conducted at 45, 90 and 180 days after discharge. Blood, stool and rectal swabs are collected at enrolment, hospital discharge and follow-up. The primary outcome is inpatient or post-discharge death. Secondary outcomes include readmission to hospital and nutritional status after discharge. Cohort analysis will identify modifiable risks, children with distinct phenotypes, relationships between factors and mechanisms underlying poor outcomes that may be targets for intervention. A nested case-control study examining infectious, immunological, metabolic, nutritional and other biological factors will be undertaken.Ethics and dissemination: This study protocol was reviewed and approved primarily by the Oxford Tropical Research Ethics Committee, and the institutional review boards of all partner sites. The study is being externally monitored. Results will be published in open access peer-reviewed scientific journals and presented to academic and policy stakeholders

Biomarkers of post-discharge mortality among children with complicated severe acute malnutrition

High mortality after discharge from hospital following acute illness has been observed among children with Severe Acute Malnutrition (SAM). However, mechanisms that may be amenable to intervention to reduce risk are unknown. We performed a nested case-control study among HIV-uninfected children aged 2-59 months treated for complicated SAM according to WHO recommendations at four Kenyan hospitals. Blood was drawn from 1778 children when clinically judged stable before discharge from hospital. Cases were children who died within 60 days. Controls were randomly selected children who survived for one year without readmission to hospital. Untargeted proteomics, total protein, cytokines and chemokines, and leptin were assayed in plasma and corresponding biological processes determined. Among 121 cases and 120 controls, increased levels of calprotectin, von Willebrand factor, angiotensinogen, IL8, IL15, IP10, TNF alpha, and decreased levels of leptin, heparin cofactor 2, and serum paraoxonase were associated with mortality after adjusting for possible confounders. Acute phase responses, cellular responses to lipopolysaccharide, neutrophil responses to bacteria, and endothelial responses were enriched among cases. Among apparently clinically stable children with SAM, a sepsis-like profile is associated with subsequent death. This may be due to ongoing bacterial infection, translocated bacterial products or deranged immune response during nutritional recovery

Cessation of exclusive breastfeeding and seasonality, but not small intestinal bacterial overgrowth, are associated with environmental enteric dysfunction: A birth cohort study amongst infants in rural Kenya.

Background: Environmental Enteric Dysfunction (EED) is a chronic intestinal inflammatory disorder of unclear aetiology prevalent amongst children in low-income settings and associated with stunting. We aimed to characterise development of EED and its putative risk factors amongst rural Kenyan infants. Methods: In a birth cohort study in Junju, rural coastal Kenya, between August 2015 and January 2017, 100 infants were each followed for nine months. Breastfeeding status was recorded weekly and anthropometry monthly. Acute illnesses and antibiotics were captured by active and passive surveillance. Intestinal function and small intestinal bacterial overgrowth (SIBO) were assessed by monthly urinary lactulose mannitol (LM) and breath hydrogen tests. Faecal alpha-1-antitrypsin, myeloperoxidase and neopterin were measured as EED biomarkers, and microbiota composition assessed by 16S sequencing. Findings: Twenty nine of the 88 participants (33%) that underwent length measurement at nine months of age were stunted (length-for-age Z score <-2). During the rainy season, linear growth was slower and LM ratio was higher. In multivariable models, LM ratio, myeloperoxidase and neopterin increased after cessation of continuous-since-birth exclusive breastfeeding. For LM ratio this only occurred during the rainy season. EED markers were not associated with antibiotics, acute illnesses, SIBO, or gut microbiota diversity. Microbiota diversified with age and was not strongly associated with complementary food introduction or linear growth impairment. Interpretation: Our data suggest that intensified promotion of uninterrupted exclusive breastfeeding amongst infants under six months during the rainy season, where rainfall is seasonal, may help prevent EED. Our findings also suggest that therapeutic strategies directed towards SIBO are unlikely to impact on EED in this setting. However, further development of non-invasive diagnostic methods for SIBO is required. Funding: This research was funded in part by the Wellcome Trust (Research Training Fellowship to RJC (103376/Z/13/Z)). EPKP was supported by the MRC/DfID Newton Fund (MR/N006259/1). JAB was supported by the MRC/DFiD/Wellcome Trust Joint Global Health Trials scheme (MR/M007367/1) and the Bill & Melinda Gates Foundation (OPP1131320). HHU was supported by the NIHR Oxford Biomedical Research Centre (IS-BRC-1215-20008)

ILC3 function as a double-edged sword in inflammatory bowel diseases

Inflammatory bowel diseases (IBD), composed mainly of Crohn’s disease (CD) and ulcerative colitis (UC), are strongly implicated in the development of intestinal inflammation lesions. Its exact etiology and pathogenesis are still undetermined. Recently accumulating evidence supports that group 3 innate lymphoid cells (ILC3) are responsible for gastrointestinal mucosal homeostasis through moderate generation of IL-22, IL-17, and GM-CSF in the physiological state. ILC3 contribute to the progression and aggravation of IBD while both IL-22 and IL-17, along with IFN-γ, are overexpressed by the dysregulation of NCR− ILC3 or NCR+ ILC3 function and the bias of NCR+ ILC3 towards ILC1 as well as regulatory ILC dysfunction in the pathological state. Herein, we feature the group 3 innate lymphoid cells’ development, biological function, maintenance of gut homeostasis, mediation of IBD occurrence, and potential application to IBD therapy

Analysis of the immune evasion mechanisms of varicella zoster virus

Varicella zoster virus (VZV) is an alpha herpes virus that causes primary infection with varicella (chicken pox), establishes latency in ganglia and may later reactivate as herpes zoster (shingles). Innate immune effectors are thought to control initial viral replication, but it is the adaptive immune system, involving T cells that mediates eventual control of viraemia and the associated clinical disease. Although both CD4+ and CD8+ T cells mediate viral clearance during acute illness, memory responses are dominated by CD4+ T cells. We tested the hypothesis that the paucity in memory CD8+ T cell effectors is partly attributed to immune evasion mechanisms that are mounted by VZV. We confirmed that VZV readily down regulates cell surface HLA-A and HLA-C but spares HLA-B onVZV infected keratinocytes and VZV infected Mewo cells. Analysis of intracellular HLA protein expression and gene transcription showed global down regulation of all HLA subtypes. Further analysis showed that VZV inhibits IFN-γ mediated up regulation of HLA expression and augments IFN-γ mediated up regulation of HLA-E and CD71 expression. Furthermore, we show that acute VZV infection lowers the frequency of circulating peripheral blood myeloid dendritic cells (mDC), reduces the expression of the DC activation marker HLA-DR and impairs inflammatory cytokine secretion in blood DC populations. Inhibition of DC cytokine secretion was found to be dependent on viral replication as irradiated virus resulted only in mild inhibition of IFN-α and TNF-α secretion. Lastly, we observed that VZV infection results in increased expression of host peptides, including MHC derived leader sequences that potentially bind to HLA-E. Cell surface HLA-E is known to be a ligand for the natural killer (NK) cell inhibitory receptor CD94/ NKG2A identifying a novel mechanism of viral immune escape from NK cell surveillance. In conclusion, our data reiterates the fact that VZV targets different aspects of antigen presentation to evade the immune system with implications for pathogenesis and approaches to improved vaccination and treatment.</p

Analysis of the immune evasion mechanisms of varicella zoster virus

Varicella zoster virus (VZV) is an alpha herpes virus that causes primary infection with varicella (chicken pox), establishes latency in ganglia and may later reactivate as herpes zoster (shingles). Innate immune effectors are thought to control initial viral replication, but it is the adaptive immune system, involving T cells that mediates eventual control of viraemia and the associated clinical disease. Although both CD4+ and CD8+ T cells mediate viral clearance during acute illness, memory responses are dominated by CD4+ T cells. We tested the hypothesis that the paucity in memory CD8+ T cell effectors is partly attributed to immune evasion mechanisms that are mounted by VZV. We confirmed that VZV readily down regulates cell surface HLA-A and HLA-C but spares HLA-B onVZV infected keratinocytes and VZV infected Mewo cells. Analysis of intracellular HLA protein expression and gene transcription showed global down regulation of all HLA subtypes. Further analysis showed that VZV inhibits IFN-γ mediated up regulation of HLA expression and augments IFN-γ mediated up regulation of HLA-E and CD71 expression. Furthermore, we show that acute VZV infection lowers the frequency of circulating peripheral blood myeloid dendritic cells (mDC), reduces the expression of the DC activation marker HLA-DR and impairs inflammatory cytokine secretion in blood DC populations. Inhibition of DC cytokine secretion was found to be dependent on viral replication as irradiated virus resulted only in mild inhibition of IFN-α and TNF-α secretion. Lastly, we observed that VZV infection results in increased expression of host peptides, including MHC derived leader sequences that potentially bind to HLA-E. Cell surface HLA-E is known to be a ligand for the natural killer (NK) cell inhibitory receptor CD94/ NKG2A identifying a novel mechanism of viral immune escape from NK cell surveillance. In conclusion, our data reiterates the fact that VZV targets different aspects of antigen presentation to evade the immune system with implications for pathogenesis and approaches to improved vaccination and treatment.This thesis is not currently available on ORA

Plasma calprotectin as a biomarker of mortality at antiretroviral treatment initiation in advanced HIV – pilot study

Background: Background: In advanced HIV, significant mortality occurs soon after starting antiretroviral treatment (ART) in low- and middle-incomes countries. Calprotectin is a biomarker of innate response to infection and inflammatory conditions. We examined the association between plasma calprotectin collected before ART treatment and mortality among individuals with advanced HIV. Methods: We conducted a pilot case-cohort study among HIV infected adults and adolescents over 13 years old with CD4+ Results: Baseline median (IQR) plasma calprotectin was 6.82 (2.65–12.5) µg/ml in cases (n=39) and 5.01 (1.92–11.5) µg/ml in non-cases (n=58). Baseline calprotectin was associated with age, neutrophil count and the presence of cough, but not other measured indicators of infection. In adjusted multivariable models, baseline calprotectin was associated with subsequent mortality: HR 1.64 (95% CI 1.11 - 2.42) and HR 2.77 (95% CI 1.58 - 4.88) for deaths during the first twenty-four and four weeks respectively. Calprotectin levels fell between baseline and 4 weeks among both cases and non-cases irrespective of randomised interventions. Conclusions: Among individuals with advanced HIV starting ART in Kenya, plasma calprotectin may have potential as a biomarker of early mortality. Validation in larger studies, comparison with other biomarkers and investigation of the sources of infection and inflammation are warranted.</p