Short-term and medium-term clinical outcomes of multisystem inflammatory syndrome in children: a prospective observational cohort study.

BACKGROUND: Even though the incidence of Multisystem Inflammatory Syndrome in children (MIS-C) is decreasing cases are still reported across the world. Studying the consequences of MIS-C enhances our understanding of the disease's prognosis. The objective of this study was to assess short- and medium-term clinical outcomes of MIS-C. METHODS: Prospective observational cohort study at Municipal Children's Hospital Morozovskaya, Moscow, Russia. All children meeting the Royal College of Paediatrics and Child Health (RCPCH), Centers for Disease Control and Prevention (CDC), or the World Health Organization (WHO) MIS-C case definition admitted to the hospital between 17 May and 26 October 2020 were included in the study. All survivors were invited to attend a clinic at 2 and 6 weeks after hospital discharge. RESULTS: 37 children median age 6 years (interquartile range [IQR] 3.3-9.4), 59.5% (22/37) boys were included in the study. 48.6% (18/37) of patients required ICU care. One child died. All children had increased levels of systemic inflammatory markers during the acute event. Echocardiographic investigations identified abnormal findings in 35.1% (13/37) of children. 5.6% (2/36) of children were presenting with any symptoms six weeks after discharge. By six weeks the inflammatory markers were within the reference norms in all children. The echocardiographic evaluation showed persistent coronary dilatation in one child. CONCLUSIONS: Despite the severity of their acute MIS-C, the majority of children in our cohort fully recovered with none having elevated laboratory markers of inflammation at 6 weeks, few (< 10%) reporting persistent symptoms at 6 weeks, and only one with persistent echocardiographic abnormalities.

A multi-platform approach to identify a blood-based host protein signature for distinguishing between bacterial and viral infections in febrile children (PERFORM): a multi-cohort machine learning study.

BACKGROUND: Differentiating between self-resolving viral infections and bacterial infections in children who are febrile is a common challenge, causing difficulties in identifying which individuals require antibiotics. Studying the host response to infection can provide useful insights and can lead to the identification of biomarkers of infection with diagnostic potential. This study aimed to identify host protein biomarkers for future development into an accurate, rapid point-of-care test that can distinguish between bacterial and viral infections, by recruiting children presenting to health-care settings with fever or a history of fever in the previous 72 h. METHODS: In this multi-cohort machine learning study, patient data were taken from EUCLIDS, the Swiss Pediatric Sepsis study, the GENDRES study, and the PERFORM study, which were all based in Europe. We generated three high-dimensional proteomic datasets (SomaScan and two via liquid chromatography tandem mass spectrometry, referred to as MS-A and MS-B) using targeted and untargeted platforms (SomaScan and liquid chromatography mass spectrometry). Protein biomarkers were then shortlisted using differential abundance analysis, feature selection using forward selection-partial least squares (FS-PLS; 100 iterations), along with a literature search. Identified proteins were tested with Luminex and ELISA and iterative FS-PLS was done again (25 iterations) on the Luminex results alone, and the Luminex and ELISA results together. A sparse protein signature for distinguishing between bacterial and viral infections was identified from the selected proteins. The performance of this signature was finally tested using Luminex assays and by calculating disease risk scores. FINDINGS: 376 children provided serum or plasma samples for use in the discovery of protein biomarkers. 79 serum samples were collected for the generation of the SomaScan dataset, 147 plasma samples for the MS-A dataset, and 150 plasma samples for the MS-B dataset. Differential abundance analysis, and the first round of feature selection using FS-PLS identified 35 protein biomarker candidates, of which 13 had commercial ELISA or Luminex tests available. 16 proteins with ELISA or Luminex tests available were identified by literature review. Further evaluation via Luminex and ELISA and the second round of feature selection using FS-PLS revealed a six-protein signature: three of the included proteins are elevated in bacterial infections (SELE, NGAL, and IFN-γ), and three are elevated in viral infections (IL18, NCAM1, and LG3BP). Performance testing of the signature using Luminex assays revealed area under the receiver operating characteristic curve values between 89·4% and 93·6%. INTERPRETATION: This study has led to the identification of a protein signature that could be ultimately developed into a blood-based point-of-care diagnostic test for rapidly diagnosing bacterial and viral infections in febrile children. Such a test has the potential to greatly improve care of children who are febrile, ensuring that the correct individuals receive antibiotics. FUNDING: European Union's Horizon 2020 research and innovation programme, the European Union's Seventh Framework Programme (EUCLIDS), Imperial Biomedical Research Centre of the National Institute for Health Research, the Wellcome Trust and Medical Research Foundation, Instituto de Salud Carlos III, Consorcio Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, Grupos de Refeencia Competitiva, Swiss State Secretariat for Education, Research and Innovation.

Immunomodulatory Therapy for MIS-C.

CONTEXT: Studies comparing initial therapy for multisystem inflammatory syndrome in children (MIS-C) provided conflicting results. OBJECTIVE: To compare outcomes in MIS-C patients treated with intravenous immunoglobulin (IVIG), glucocorticoids, or the combination thereof. DATA SOURCES: Medline, Embase, CENTRAL and WOS, from January 2020 to February 2022. STUDY SELECTION: Randomized or observational comparative studies including MIS-C patients <21 years. DATA EXTRACTION: Two reviewers independently selected studies and obtained individual participant data. The main outcome was cardiovascular dysfunction (CD), defined as left ventricular ejection fraction < 55% or vasopressor requirement ≥ day 2 of initial therapy, analyzed with a propensity score-matched analysis. RESULTS: Of 2635 studies identified, 3 nonrandomized cohorts were included. The meta-analysis included 958 children. IVIG plus glucocorticoids group as compared with IVIG alone had improved CD (odds ratio [OR] 0.62 [0.42-0.91]). Glucocorticoids alone group as compared with IVIG alone did not have improved CD (OR 0.57 [0.31-1.05]). Glucocorticoids alone group as compared with IVIG plus glucocorticoids did not have improved CD (OR 0.67 [0.24-1.86]). Secondary analyses found better outcomes associated with IVIG plus glucocorticoids compared with glucocorticoids alone (fever ≥ day 2, need for secondary therapies) and better outcomes associated with glucocorticoids alone compared with IVIG alone (left ventricular ejection fraction < 55% ≥ day 2). LIMITATIONS: Nonrandomized nature of included studies. CONCLUSIONS: In a meta-analysis of MIS-C patients, IVIG plus glucocorticoids was associated with improved CD compared with IVIG alone. Glucocorticoids alone was not associated with improved CD compared with IVIG alone or IVIG plus glucocorticoids.

Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study.

Background: Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. Methods: The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. Findings: We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2-11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75-1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58-1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91-1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70-1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11-0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50-0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38-0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45-0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. Interpretation: Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. Funding: Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health.

Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatory phenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

The immunopathogenesis of SARS-CoV-2 infection in children: diagnostics, treatment and prevention.

Symptoms and outcomes for paediatric COVID-19 differ vastly from those for adults, with much lower morbidity and mortality. Immunopathogenesis drives severe outcomes in adults, and it is likely that age-related differences in both the innate and specific immune responses underlie much of the variation. Understanding the protective features of the paediatric immune system may be crucial to better elucidate disease severity in adult COVID-19 and may pave the way for novel therapeutic approaches. However, as well as uncommon cases of severe paediatric acute COVID-19, there have been children who have presented with delayed multisystem inflammation, including cardiac, gastrointestinal, skin, mucosa and central nervous system involvement. The occurrence of coronary artery aneurysms has drawn comparisons with Kawasaki Disease, but similarities with the inflammatory phase of adult acute COVID-19 have also been drawn. In this review, we summarise findings from studies investigating pre-existing immunity, cytokine profiles, innate, B-cell, antibody, T-cell and vaccine responses in children with acute COVID-19 and multisystem inflammation, compared with COVID-19 adults and controls. We further consider the relevance to therapeutics in the context of limited evidence in children and highlight key questions to be answered about the immune response of children to SARS-CoV-2.

Treatment of Multisystem Inflammatory Syndrome in Children: Understanding Differences in Results of Comparative Effectiveness Studies.

OBJECTIVE: Two cohort studies in patients with multisystem inflammatory syndrome in children (MIS-C) demonstrated contrasting results regarding the benefit of initial immunomodulatory treatment with intravenous immunoglobulin (IVIG) alone versus IVIG and glucocorticoids. We sought to determine whether application of different MIS-C definitions and differing disease severity between cohorts underlay discrepant results. METHODS: The Overcoming COVID-19 Public Health Surveillance Registry (OC-19) included patients meeting the US Centers for Disease Control and Prevention (CDC) MIS-C definition, whereas the Best Available Treatment Study (BATS) applied the World Health Organization (WHO) definition. We applied the WHO definition to the OC-19 cohort and the CDC definition to the BATS cohort and determined the proportion that did not meet the alternate definition. We compared illness severity indicators between cohorts. RESULTS: Of 349 OC-19 patients, 9.5% did not meet the WHO definition. Of 350 BATS patients, 10.3% did not meet the CDC definition. Most organ system involvement was similar between the cohorts, but more OC-19 patients had WHO-defined cardiac involvement (87.1% vs 79.4%, P = 0.008). OC-19 patients were more often admitted to intensive care (61.0% vs 44.8%, P < 0.001) and more often received vasopressors or inotropes (39.5% vs 22.9%, P < 0.001) before immunomodulatory treatment. CONCLUSION: Greater illness severity and cardiovascular involvement in the OC-19 cohort compared with the BATS cohort, and not use of different MIS-C case definitions, may have contributed to differing study conclusions about optimal initial treatment for MIS-C. Disease severity should be considered in future MIS-C study designs and treatment recommendations to identify patients who would benefit from aggressive immunomodulatory treatment.

Kawasaki Disease Patient Stratification and Pathway Analysis Based on Host Transcriptomic and Proteomic Profiles.

The aetiology of Kawasaki disease (KD), an acute inflammatory disorder of childhood, remains unknown despite various triggers of KD having been proposed. Host 'omic profiles offer insights into the host response to infection and inflammation, with the interrogation of multiple 'omic levels in parallel providing a more comprehensive picture. We used differential abundance analysis, pathway analysis, clustering, and classification techniques to explore whether the host response in KD is more similar to the response to bacterial or viral infections at the transcriptomic and proteomic levels through comparison of 'omic profiles from children with KD to those with bacterial and viral infections. Pathways activated in patients with KD included those involved in anti-viral and anti-bacterial responses. Unsupervised clustering showed that the majority of KD patients clustered with bacterial patients on both 'omic levels, whilst application of diagnostic signatures specific for bacterial and viral infections revealed that many transcriptomic KD samples had low probabilities of having bacterial or viral infections, suggesting that KD may be triggered by a different process not typical of either common bacterial or viral infections. Clustering based on the transcriptomic and proteomic responses during KD revealed three clusters of KD patients on both 'omic levels, suggesting heterogeneity within the inflammatory response during KD. The observed heterogeneity may reflect differences in the host response to a common trigger, or variation dependent on different triggers of the condition.

Treatment of Multisystem Inflammatory Syndrome in Children.

BACKGROUND: Evidence is urgently needed to support treatment decisions for children with multisystem inflammatory syndrome (MIS-C) associated with severe acute respiratory syndrome coronavirus 2. METHODS: We performed an international observational cohort study of clinical and outcome data regarding suspected MIS-C that had been uploaded by physicians onto a Web-based database. We used inverse-probability weighting and generalized linear models to evaluate intravenous immune globulin (IVIG) as a reference, as compared with IVIG plus glucocorticoids and glucocorticoids alone. There were two primary outcomes: the first was a composite of inotropic support or mechanical ventilation by day 2 or later or death; the second was a reduction in disease severity on an ordinal scale by day 2. Secondary outcomes included treatment escalation and the time until a reduction in organ failure and inflammation. RESULTS: Data were available regarding the course of treatment for 614 children from 32 countries from June 2020 through February 2021; 490 met the World Health Organization criteria for MIS-C. Of the 614 children with suspected MIS-C, 246 received primary treatment with IVIG alone, 208 with IVIG plus glucocorticoids, and 99 with glucocorticoids alone; 22 children received other treatment combinations, including biologic agents, and 39 received no immunomodulatory therapy. Receipt of inotropic or ventilatory support or death occurred in 56 patients who received IVIG plus glucocorticoids (adjusted odds ratio for the comparison with IVIG alone, 0.77; 95% confidence interval [CI], 0.33 to 1.82) and in 17 patients who received glucocorticoids alone (adjusted odds ratio, 0.54; 95% CI, 0.22 to 1.33). The adjusted odds ratios for a reduction in disease severity were similar in the two groups, as compared with IVIG alone (0.90 for IVIG plus glucocorticoids and 0.93 for glucocorticoids alone). The time until a reduction in disease severity was similar in the three groups. CONCLUSIONS: We found no evidence that recovery from MIS-C differed after primary treatment with IVIG alone, IVIG plus glucocorticoids, or glucocorticoids alone, although significant differences may emerge as more data accrue. (Funded by the European Union's Horizon 2020 Program and others; BATS ISRCTN number, ISRCTN69546370.).

What is proteomics?

Proteomics is the study of a large number of proteins in biological systems. We aim to introduce the complex field to paediatricians and present some recent examples of applications to paediatric problems. Various approaches have been used to study proteomes. The current mainstay is tandem mass spectrometry of enzymatically digested proteins ('bottom-up proteomics'), and we describe the experimental and computational approach further. Proteomics can offer advantages over transcriptomics by giving direct information about proteins rather than RNA; however, typically data are obtained at lower depth and the confident identification of mass spectra can be challenging. Proteomics frequently complements transcriptomics and other -omics. Used effectively, proteomics offers promise to help answer important clinical and biological questions.

A proteomics-based method for identifying antigens within immune complexes.

A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue.

A recent study reported that increasing host DNA abundance and reducing read depth impairs the sensitivity of detection of low-abundance micro-organisms by shotgun metagenomics. The authors used DNA from a synthetic bacterial community with abundances varying across several orders of magnitude and added varying proportions of host DNA. However, the use of a marker-gene-based abundance estimation tool (MetaPhlAn2) requires considerable depth to detect marker genes from low-abundance organisms. Here, we reanalyse the deposited data, and place the study in the broader context of low microbial biomass metagenomics. We opted for a fast and sensitive read binning tool (Kraken 2) with abundance estimates from Bracken. With this approach all organisms are detected even when the sample comprises 99 % host DNA and similarly accurate abundance estimates are provided (mean squared error 0.45 vs. 0.3 in the original study). We show that off-target genera, whether contaminants or misidentified reads, come to represent over 10 % of reads when the sample is 99 % host DNA and exceed counts of many target genera. Therefore, we applied Decontam, a contaminant detection tool, which was able to remove 61 % of off-target species and 79 % of off-target reads. We conclude that read binning tools can remain sensitive to low-abundance organisms even with high host DNA content, but even low levels of contamination pose a significant problem due to low microbial biomass. Analytical mitigations are available, such as Decontam, although steps to reduce contamination are critical.

When do co-infections matter?

PURPOSE OF REVIEW: Advances in diagnostic methods mean that co-infections are increasingly being detected in clinical practice, yet their significance is not always obvious. In parallel, basic science studies are increasingly investigating interactions between pathogens to try to explain real-life observations and elucidate biological mechanisms. RECENT FINDINGS: Co-infections may be insignificant, detrimental, or even beneficial, and these outcomes can occur through multiple levels of interactions which include modulation of the host response, altering the performance of diagnostic tests, and drug-drug interactions during treatment. The harmful effects of chronic co-infections such as tuberculosis or Hepatitis B and C in association with HIV are well established, and recent studies have focussed on strategies to mitigate these effects. However, consequences of many acute co-infections are much less certain, and recent conflicting findings simply highlight many of the challenges of studying naturally acquired infections in humans. SUMMARY: Tackling these challenges, using animal models, or careful prospective studies in humans may prove to be worthwhile. There are already tantalizing examples where identification and treatment of relevant co-infections seems to hold promise for improved health outcomes.

Determinants of Carboxyhemoglobin Levels and Relationship with Sepsis in a Retrospective Cohort of Preterm Neonates.

Carboxyhemoglobin levels in blood reflect endogenous carbon monoxide production and are often measured during routine blood gas analysis. Endogenous carbon monoxide production has been reported to be increased during sepsis, but carboxyhemoglobin levels have not been thoroughly evaluated as a biomarker of sepsis. We sought to determine whether carboxyhemoglobin levels were elevated during sepsis in a high risk population of premature neonates. We conducted a retrospective cohort study of 30 infants in two neonatal intensive care units using electronic medical and laboratory records. The majority of infants were extremely premature and extremely low birth weight, and 25 had at least one episode of sepsis. We collected all carboxyhemoglobin measurements during their in-patient stay and examined the relationship between carboxyhemoglobin and a variety of clinical and laboratory parameters, in addition to the presence or absence of sepsis, using linear mixed-effect models. We found that postnatal age had the most significant effect on carboxyhemoglobin levels, and other significant associations were identified with gestational age, hemoglobin concentration, oxyhemoglobin saturation, and blood pH. Accounting for these covariates, there was no significant relationship between the onset of sepsis and carboxyhemoglobin levels. Our results show that carboxyhemoglobin is unlikely to be a clinically useful biomarker of sepsis in premature infants, and raise a note of caution about factors which may confound the use of carbon monoxide as a clinical biomarker for other disease processes such as hemolysis.

Hypervitaminosis A is prevalent in children with CKD and contributes to hypercalcemia.

BACKGROUND: Vitamin A accumulates in renal failure, but the prevalence of hypervitaminosis A in children with predialysis chronic kidney disease (CKD) is not known. Hypervitaminosis A has been associated with hypercalcemia. In this study we compared dietary vitamin A intake with serum retinoid levels and their associations with hypercalcemia. METHODS: We studied the relationship between vitamin A intake, serum retinoid levels, and serum calcium in 105 children with CKD stages 2-5 on dialysis and posttransplant. Serum retinoid measures included retinol (ROH), its active retinoic acid (RA) metabolites [all-trans RA (at-RA) and 13-cis RA] and carrier proteins [retinol-binding protein-4 (RBP4) and transthyretin (TTR)]. Dietary vitamin A intake was assessed using a food diary. RESULTS: Twenty-five children were in CKD 2-3, 35 in CKD 4-5, 23 on dialysis and 22 posttransplant; 53 % had vitamin A intake above the Reference Nutrient Intake (RNI) value. Children receiving supplemental feeds compared with diet alone had higher vitamin A intake (p = 0.02) and higher serum ROH (p < 0.001). Notably, increased ROH was seen as early as CKD stage 2. For every 10 ml/min/1.73 m(2) fall in estimated glomerular filtration rate (eGFR), there was a 13 % increase in ROH. RBP4 levels were increased in CKD 3-5 and dialysis patients. The lowest ratios of ROH:RBP4 were seen in dialysis compared with CKD 2-3 (p = 0.03), suggesting a relative increase in circulating RBP4. Serum ROH, RBP4 and at-RA were associated with serum calcium. On multivariable analysis RBP4 levels and alfacalcidol dose were significant predictors of serum calcium (model R (2) 32 %) in dialysis patients. CONCLUSIONS: Hypervitaminosis A is seen in early CKD, with highest levels in children on supplemental feeds compared with diet alone. Serum retinoid levels significantly predict hypercalcemia.