Prothrombotic autoantibodies targeting platelet factor 4/polyanion are associated with pediatric cerebral malaria.

BACKGROUNDFeatures of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM.METHODSPlasma from children with uncomplicated malaria (UM) (n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-platelet factor 4/polyanion (anti-PF4/P), anti-phospholipid, anti-phosphatidylserine, anti-myeloperoxidase, anti-proteinase 3, anti-dsDNA, anti-ß-2-glycoprotein I, and anti-cardiolipin. Plasma samples from individuals with nonmalarial coma (NMC) (n = 49) and healthy controls (HCs) (n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses.RESULTSMedian anti-PF4/P and anti-PS IgG levels were elevated in individuals with malaria infection relative to levels in HCs (P < 0.001) and patients with NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in children with CM (median = 0.27, IQR: 0.19-0.41) compared with those with UM (median = 0.19, IQR: 0.14-0.22, P < 0.0001). Anti-PS IgG levels did not differ between patients with UM and those with CM (P = 0.39). When patients with CM were stratified by malaria retinopathy (Ret) status, the levels of anti-PF4/P IgG correlated negatively with the peripheral platelet count in patients with Ret+ CM (Spearman's rho [Rs] = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02-275, P = 0.048). Plasma from patients with CM induced greater platelet activation in an ex vivo assay relative to plasma from patients with UM (P = 0.02), and the observed platelet activation was associated with anti-PF4/P IgG levels (Rs= 0.293, P = 0.035).CONCLUSIONSThrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.

Cerebral Metabolic Crisis in Pediatric Cerebral Malaria.

Cerebral metabolic energy crisis (CMEC), often defined as a cerebrospinal fluid (CSF) lactate: pyruvate ratio (LPR) >40, occurs in various diseases and is associated with poor neurologic outcomes. Cerebral malaria (CM) causes significant mortality and neurodisability in children worldwide. Multiple factors that could lead to CMEC are plausible in these patients, but its frequency has not been explored. Fifty-three children with CM were enrolled and underwent analysis of CSF lactate and pyruvate levels. All 53 patients met criteria for a CMEC (median CSF LPR of 72.9 [interquartile range [IQR]: 58.5-93.3]). Half of children met criteria for an ischemic CMEC (median LPR of 85 [IQR: 73-184]) and half met criteria for a nonischemic CMEC (median LPR of 60 [IQR: 54-79]. Children also underwent transcranial doppler ultrasound investigation. Cerebral blood flow velocities were more likely to meet diagnostic criteria for low flow (<2 standard deviation from normal) or vasospasm in children with an ischemic CMEC (73%) than in children with a nonischemic CMEC (20%, p = 0.04). Children with an ischemic CMEC had poorer outcomes (pediatric cerebral performance category of 3-6) than those with a nonischemic CMEC (46 vs. 22%, p = 0.03). CMEC was ubiquitous in this patient population and the processes underlying the two subtypes (ischemic and nonischemic) may represent targets for future adjunctive therapies.

Exploring Nurses' Perceptions of Specialty Certification.

BACKGROUND: The strategic nursing plan for a large Midwestern health care system includes achievement of the American Nurses Credentialing Center's Magnet® recognition for all their hospitals. This study explored the barriers to and perceived value of certification among nurses employed by the same health care system across eight facilities in a metropolitan region. One of these facilities holds Magnet® recognition. METHOD: Subjects were recruited by direct email to collect demographic data and complete a barrier to certification survey and the Perceived Value of Certification Tool-12. RESULTS: There were statistically significant differences in the perception that lack of institutional support is a barrier to certification both in aggregate and when comparing the Magnet® hospital with the others. CONCLUSION: As an initial exploration into barriers and perceived value, this study provides data for this health care system to plan strategies to promote certification. This study also serves as an exemplar for other organizations considering evaluation of certification barriers and value among their nursing staff. [J Contin Educ Nurs. 2023;54(12):567-573.].

Aetiology and outcome of non-traumatic coma in African children: protocol for a systematic review and meta-analysis.

BACKGROUND: Non-traumatic coma is a common acute childhood presentation to healthcare facilities in Africa and is associated with high morbidity and mortality. Historically, the majority of cases were attributed to cerebral malaria (CM). With the recent drastic reduction in malaria incidence, non-malarial coma is becoming a larger proportion of cases and determining the aetiology is diagnostically challenging, particularly in resource-limited settings. The purpose of this study will be to evaluate the aetiology and prognosis of non-traumatic coma in African children. METHODS: With no date restrictions, systematic searches of MEDLINE, Embase, and Scopus will identify prospective and retrospective studies (including randomised controlled trials, cluster randomised trials, cohort studies, cross-sectional, and case-control studies) recruiting children (1 month-16 years) with non-traumatic coma (defined by Blantyre Coma Score ≤ 2 or comparable alternative) from any African country. Disease-specific studies will be included if coma is associated and reported. The primary outcome is to determine the aetiology (infectious and non-infectious) of non-traumatic coma in African children, with pooled prevalence estimates of causes (e.g., malaria). Secondary outcomes are to determine overall estimates of morbidity and mortality of all-cause non-traumatic coma and disease-specific states of non-traumatic coma, where available. Random effects meta-analysis will summarise aetiology data and in-hospital and post-discharge mortality. Heterogeneity will be quantified with τ2, I2, and Cochran's Q test. DISCUSSION: This systematic review will provide a summary of the best available evidence on the aetiology and outcome of non-traumatic coma in African children. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020141937.

Spectrum, risk factors and outcomes of neurological and psychiatric complications of COVID-19: a UK-wide cross-sectional surveillance study.

SARS-CoV-2 is associated with new-onset neurological and psychiatric conditions. Detailed clinical data, including factors associated with recovery, are lacking, hampering prediction modelling and targeted therapeutic interventions. In a UK-wide cross-sectional surveillance study of adult hospitalized patients during the first COVID-19 wave, with multi-professional input from general and sub-specialty neurologists, psychiatrists, stroke physicians, and intensivists, we captured detailed data on demographics, risk factors, pre-COVID-19 Rockwood frailty score, comorbidities, neurological presentation and outcome. A priori clinical case definitions were used, with cross-specialty independent adjudication for discrepant cases. Multivariable logistic regression was performed using demographic and clinical variables, to determine the factors associated with outcome. A total of 267 cases were included. Cerebrovascular events were most frequently reported (131, 49%), followed by other central disorders (95, 36%) including delirium (28, 11%), central inflammatory (25, 9%), psychiatric (25, 9%), and other encephalopathies (17, 7%), including a severe encephalopathy (n = 13) not meeting delirium criteria; and peripheral nerve disorders (41, 15%). Those with the severe encephalopathy, in comparison to delirium, were younger, had higher rates of admission to intensive care and a longer duration of ventilation. Compared to normative data during the equivalent time period prior to the pandemic, cases of stroke in association with COVID-19 were younger and had a greater number of conventional, modifiable cerebrovascular risk factors. Twenty-seven per cent of strokes occurred in patients <60 years. Relative to those >60 years old, the younger stroke patients presented with delayed onset from respiratory symptoms, higher rates of multi-vessel occlusion (31%) and systemic thrombotic events. Clinical outcomes varied between disease groups, with cerebrovascular disease conferring the worst prognosis, but this effect was less marked than the pre-morbid factors of older age and a higher pre-COVID-19 frailty score, and a high admission white cell count, which were independently associated with a poor outcome. In summary, this study describes the spectrum of neurological and psychiatric conditions associated with COVID-19. In addition, we identify a severe COVID-19 encephalopathy atypical for delirium, and a phenotype of COVID-19 associated stroke in younger adults with a tendency for multiple infarcts and systemic thromboses. These clinical data will be useful to inform mechanistic studies and stratification of patients in clinical trials.

Neurological manifestations of SARS-CoV-2 infection in hospitalised children and adolescents in the UK: a prospective national cohort study.

BACKGROUND: The spectrum of neurological and psychiatric complications associated with paediatric SARS-CoV-2 infection is poorly understood. We aimed to analyse the range and prevalence of these complications in hospitalised children and adolescents. METHODS: We did a prospective national cohort study in the UK using an online network of secure rapid-response notification portals established by the CoroNerve study group. Paediatric neurologists were invited to notify any children and adolescents (age <18 years) admitted to hospital with neurological or psychiatric disorders in whom they considered SARS-CoV-2 infection to be relevant to the presentation. Patients were excluded if they did not have a neurological consultation or neurological investigations or both, or did not meet the definition for confirmed SARS-CoV-2 infection (a positive PCR of respiratory or spinal fluid samples, serology for anti-SARS-CoV-2 IgG, or both), or the Royal College of Paediatrics and Child Health criteria for paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS). Individuals were classified as having either a primary neurological disorder associated with COVID-19 (COVID-19 neurology group) or PIMS-TS with neurological features (PIMS-TS neurology group). The denominator of all hospitalised children and adolescents with COVID-19 was collated from National Health Service England data. FINDINGS: Between April 2, 2020, and Feb 1, 2021, 52 cases were identified; in England, there were 51 cases among 1334 children and adolescents hospitalised with COVID-19, giving an estimated prevalence of 3·8 (95% CI 2·9-5·0) cases per 100 paediatric patients. 22 (42%) patients were female and 30 (58%) were male; the median age was 9 years (range 1-17). 36 (69%) patients were Black or Asian, 16 (31%) were White. 27 (52%) of 52 patients were classified into the COVID-19 neurology group and 25 (48%) were classified into the PIMS-TS neurology group. In the COVID-19 neurology group, diagnoses included status epilepticus (n=7), encephalitis (n=5), Guillain-Barré syndrome (n=5), acute demyelinating syndrome (n=3), chorea (n=2), psychosis (n=2), isolated encephalopathy (n=2), and transient ischaemic attack (n=1). The PIMS-TS neurology group more often had multiple features, which included encephalopathy (n=22 [88%]), peripheral nervous system involvement (n=10 [40%]), behavioural change (n=9 [36%]), and hallucinations at presentation (n=6 [24%]). Recognised neuroimmune disorders were more common in the COVID-19 neurology group than in the PIMS-TS neurology group (13 [48%] of 27 patients vs 1 [<1%] of 25 patients, p=0·0003). Compared with the COVID-19 neurology group, more patients in the PIMS-TS neurology group were admitted to intensive care (20 [80%] of 25 patients vs six [22%] of 27 patients, p=0·0001) and received immunomodulatory treatment (22 [88%] patients vs 12 [44%] patients, p=0·045). 17 (33%) patients (10 [37%] in the COVID-19 neurology group and 7 [28%] in the PIMS-TS neurology group) were discharged with disability; one (2%) died (who had stroke, in the PIMS-TS neurology group). INTERPRETATION: This study identified key differences between those with a primary neurological disorder versus those with PIMS-TS. Compared with patients with a primary neurological disorder, more patients with PIMS-TS needed intensive care, but outcomes were similar overall. Further studies should investigate underlying mechanisms for neurological involvement in COVID-19 and the longer-term outcomes. FUNDING: UK Research and Innovation, Medical Research Council, Wellcome Trust, National Institute for Health Research.

Severe stridor and profound weakness after cerebral malaria.

Cerebral malaria (CM) is defined by WHO as coma (Blantyre Coma Score 2 or less) in a patient with Plasmodium falciparum parasitaemia and no alternative cause of coma identified. Mortality is approximately 15%-30% in African children and up to one-third of survivors have neurological sequelae. We present a patient with severe stridor and prolonged profound weakness during an intensive care admission with CM. These complications initially presented a diagnostic dilemma in our limited resourced setting. The stridor failed to improve with empiric steroids and a subsequent opportunistic ENT consult diagnosed vocal cord paresis. The weakness was so profound that the patient was unable to lift his head during the acute illness. The child received intensive physiotherapy, and at 1-month follow-up, the stridor and weakness had resolved.

Plasma cell-free DNA predicts pediatric cerebral malaria severity.

BACKGROUNDPrediction of adverse outcomes in cerebral malaria (CM) is difficult. We hypothesized that cell-free DNA (cfDNA) levels would facilitate identification of severe and potentially fatal CM cases.METHODSIn this retrospective study, plasma from Malawian children with CM (n = 134), uncomplicated malaria (UM, n = 77), and healthy controls (HC, n = 60) was assayed for cfDNA using a fluorescence assay. Host and parasite cfDNA was measured by quantitative PCR. Immune markers were determined by ELISA, Luminex, or cytometric bead array.RESULTSTotal cfDNA increased with malaria severity (HC versus UM, P < 0.001; HC versus CM, P < 0.0001; UM versus CM, P < 0.0001), was elevated in retinopathy-positive (Ret+) CM relative to Ret- CM (7.66 versus 5.47 ng/µL, P = 0.027), and differentiated Ret+ fatal cases from survivors (AUC 0.779; P < 0.001). cfDNA levels in patients with non-malarial febrile illness (NMF, P = 0.25) and non-malarial coma (NMC, P = 0.99) were comparable with UM. Host DNA, rather than parasite DNA, was the major cfDNA contributor (UM, 268 versus 67 pg/µL; CM, 2824 versus 463 pg/µL). Host and parasite cfDNA distinguished CM by retinopathy (host, AUC 0.715, P = 0.0001; parasite, AUC 0.745, P = 0.0001), but only host cfDNA distinguished fatal cases (AUC 0.715, P = 0.0001). Total cfDNA correlated with neutrophil markers IL-8 (rs = 0.433, P < 0.0001) and myeloperoxidase (rs = 0.683, P < 0.0001).CONCLUSIONQuantifying plasma cfDNA is a simple assay useful in identifying children at risk for fatal outcome and has promise as a point-of-care assay. Elevated cfDNA suggests a link with host inflammatory pathways in fatal CM.FUNDINGNIH NCATS (AK), Burroughs-Wellcome (AK), and National Health and Medical Research Council of Australia (SJR).

Transcranial Doppler Ultrasonographic Evaluation of Cerebrovascular Abnormalities in Children With Acute Bacterial Meningitis.

Introduction: Bacterial meningitis (BM) is a global public health concern that results in significant morbidity and mortality. Cerebral arterial narrowing contributes to stroke in BM and may be amenable to intervention. However, it is difficult to diagnose in resource-limited settings where the disease is common. Methods: This was a prospective observational study from September 2015 to December 2019 in sub-Saharan Africa. Children 1 month-18 years of age with neutrophilic pleocytosis or a bacterial pathogen identified in the cerebrospinal fluid were enrolled. Transcranial Doppler ultrasound (TCD) of the middle cerebral arteries was performed daily with the aim to identify flow abnormalities consistent with vascular narrowing. Results: Forty-seven patients were analyzed. The majority had Streptococcus pneumoniae (36%) or Neisseria meningitides (36%) meningitis. Admission TCD was normal in 10 (21%). High flow with a normal pulsatility index (PI) was seen in 20 (43%) and high flow with a low PI was identified in 7 (15%). Ten (21%) had low flow. All children with a normal TCD had a good outcome. Patients with a high-risk TCD flow pattern (high flow/low PI or low flow) were more likely to have a poor outcome (82 vs. 38%, p = 0.001). Conclusions: Abnormal TCD flow patterns were common in children with BM and identified those at high risk of poor neurological outcome.

Effects of Supplemental Calcium and Vitamin D on Expression of Toll-Like Receptors and Phospho-IKKα/ß in the Normal Rectal Mucosa of Colorectal Adenoma Patients.

Chronic inflammation in the colorectum, a significant contributor to colorectal carcinogenesis, can be triggered by the activation of proinflammatory signaling pathways such as those initiated by Toll-like receptors (TLR) and nuclear factor κB (NF-κB). Although experimental evidence supports calcium and vitamin D potentially modifying these proinflammatory pathways in the colorectum, human data in these regards are scarce. We investigated supplemental calcium (1,200 mg daily) and/or vitamin D3 (1,000 IU daily) effects on inflammatory signaling pathway-related biomarkers in a subset of 105 participants from a colorectal adenoma recurrence chemoprevention clinical trial. We assessed expression of TLR4 and TLR5, which recognize the bacterial components lipopolysaccharides and flagellin, respectively, and phospho-IKKα/ß (pIKKα/ß), a biomarker of inflammation, in the normal-appearing rectal crypt epithelium and stroma using standardized, automated immunohistochemistry and quantitative image analysis. Following 1 year of treatment, TLR4, TLR5, and pIKKα/ß expression in the rectal mucosa did not statistically significantly change with vitamin D or calcium supplementation, taken alone or in combination. Several baseline participant characteristics, including body mass index, history of sessile serrated adenomas, high red/processed meat intake, and high levels of rectal epithelial cell proliferation (as measured by MIB-1/Ki-67), were associated with higher baseline expression of TLRs or pIKKα/ß. Our findings suggest that vitamin D and calcium may have no substantial effect on the investigated biomarkers. However, several modifiable lifestyle factors may be associated with TLRs and pIKKα/ß expression in the normal rectal mucosa, supporting their future investigation as potentially treatable, preneoplastic risk factors for colorectal neoplasms. Cancer Prev Res; 11(11); 707-16. ©2018 AACR.

The inter-rater reliability and prognostic value of coma scales in Nepali children with acute encephalitis syndrome.

Background Acute encephalitis syndrome (AES) is a common cause of coma in Nepali children. The Glasgow coma scale (GCS) is used to assess the level of coma in these patients and predict outcome. Alternative coma scales may have better inter-rater reliability and prognostic value in encephalitis in Nepali children, but this has not been studied. The Adelaide coma scale (ACS), Blantyre coma scale (BCS) and the Alert, Verbal, Pain, Unresponsive scale (AVPU) are alternatives to the GCS which can be used. Methods Children aged 1-14 years who presented to Kanti Children's Hospital, Kathmandu with AES between September 2010 and November 2011 were recruited. All four coma scales (GCS, ACS, BCS and AVPU) were applied on admission, 48 h later and on discharge. Inter-rater reliability (unweighted kappa) was measured for each. Correlation and agreement between total coma score and outcome (Liverpool outcome score) was measured by Spearman's rank and Bland-Altman plot. The prognostic value of coma scales alone and in combination with physiological variables was investigated in a subgroup (n = 22). A multivariable logistic regression model was fitted by backward stepwise. Results Fifty children were recruited. Inter-rater reliability using the variables scales was fair to moderate. However, the scales poorly predicted clinical outcome. Combining the scales with physiological parameters such as systolic blood pressure improved outcome prediction. Conclusion This is the first study to compare four coma scales in Nepali children with AES. The scales exhibited fair to moderate inter-rater reliability. However, the study is inadequately powered to answer the question on the relationship between coma scales and outcome. Further larger studies are required.

Preventing cerebral palsy in preterm labour: a multiorganisational quality improvement approach to the adoption and spread of magnesium sulphate for neuroprotection.

Magnesium sulphate has been demonstrated to be an effective neuroprotectant for babies delivered prematurely (under 37 weeks' gestational age). Antenatal administration reduces infant mortality and cerebral palsy (CP); however, uptake in the UK has been significantly lower than other countries. A quality improvement (QI) project (PReventing Cerebral palsy in Pre Term labour (PReCePT)) was carried out in the West of England, UK, to raise awareness of evidence and to improve the uptake of magnesium sulphate as neuroprotectant in preterm deliveries. Five National Health Service (NHS) Trusts and the West of England Academic Health Science Network participated in the QI project. The project was underpinned by a multifaceted QI approach that included: patient and clinical coproduction of resources; recruitment of clinical champions to support the local microsystems and create a stimulating/supporting environment for change; Plan, Do, Study, Act cycles; training for over 600 NHS staff and awareness raising and strategic influencing of key leaders. A baseline audit and regular measurement of the number of eligible women receiving magnesium sulphate was undertaken at each hospital site, and the overall programme was evaluated using data from an international benchmarking organisation for neonatal care outcomes-the Vermont Oxford Network. During the project 664 staff received magnesium sulphate training. The use of magnesium sulphate increased across the West of England from an average baseline of 21% over the 2 years preceding the project to 88% by the conclusion of the project. The project was also able to influence the development of a national data collection process for benchmarking the use of magnesium sulphate for neuroprotection in preterm deliveries in the U.K. PReCePT appears to have had a favourable effect on the uptake of magnesium sulphate across the West of England. The project has also provided learning about how to stimulate adoption and spread of evidence using a QI approach across a network.

Adipose Derived Cells and Tissues for Regenerative Medicine.

Adipose tissue is now recognized as a complex organ serving endocrine, immune, and metabolic functions. Adipose depots are composed of mature adipocytes as well as stromal vascular fraction (SVF) cells, a heterogeneous population of B and T lymphocytes, endothelial cells, macrophages, pericytes, smooth muscle, and stromal cells that can be isolated by enzymatic digestion. When placed into culture medium, a subset of the SVF cells can adhere to the plastic surface and expand in number. This latter population, known as "adipose-derived stromal/stem cells" (ASC), exhibits trilineage (adipo-, chondro-, osteo-) differentiation potential. There are currently more than 180 clinical studies underway worldwide exploring the regenerative medical application of SVF cells and ASC in a range of medical conditions. Plastic surgeons have a particular interest in the use of autologous fat and SVF enhanced fat for cosmetic and reconstructive surgical procedures. Orthopedic and craniofacial surgeons have begun to use ASC to treat bone and musculoskeletal defects with success. Furthermore, studies are underway to exploit the immunomodulatory function of ASC to treat immune-mediated disorders such as Crohn's disease. Indeed, it is postulated that adipose tissue and cells modulate tissue regeneration and inflammatory responses through their secretion of paracrine factors. Continued advances in this emerging field will require harmonization of international standards and guidelines defining the release criteria as well as the safety and efficacy of adipose-derived cells and tissues. Currently, there are no accepted standard guidelines for autologous fat harvesting technique, processing, or method of injection. Close collaboration between academia, industry, and regulatory authorities will be necessary to ensure that adipose-derived products are affordable and quality controlled throughout the globe.

Subdural Empyema Caused by Neisseria meningitidis: A Case Report and Review of the Literature.

Subdural empyema complicating meningococcal meningitis is rare. We describe a case and 14 previously reported cases; all had persistent fever and 12 had seizures. Initial cerebrospinal fluid showed raised protein with low glucose values. Neuroimaging confirmed the diagnosis in all cases where undertaken. Ten children had neurosurgical intervention. Clinical outcome was available in 14 cases; a full recovery was reported in 10 and 1 child died.

A Pilot Study of Obesogenic Eating Behaviors in Children With Migraine.

We studied associations between migraine severity and obesogenic eating behaviors in children with a prospective cross-sectional, clinic-based study. Migraine severity was quantified using the PedMIDAS tool and attack frequency. Eating behaviors were assessed using the Dutch Eating Behaviour Questionnaire and the Child Eating Behaviour Questionnaire. Food intake was assessed using a Food Intake Questionnaire. Statistical tests of association between eating behavior, food intake, and adiposity with migraine severity were performed. Sixty children (mean age = 10.9 years, standard deviation = 3.1; 26 males) were recruited. There was a positive correlation between the Child Eating Behaviour Questionnaire desire to drink subscale and PedMIDAS scores (r = 0.41, P = .01). Attack frequency was associated with higher intake of high fat or sugar content food and drink (r = 0.27, P = .04). No association between migraine severity and adiposity was found. Suggestion that migraine severity in children is associated with certain obesogenic eating behaviors requires further large study investigation.

Rapid Identification of Microorganisms by FilmArray Blood Culture Identification Panel Improves Clinical Management in Children.

BACKGROUND: Blood cultures are a common investigation for children admitted to hospital. In routine practice, it takes at least 24 hours to identify an organism as a contaminant or clinically significant. FilmArray Blood Culture Identification Panel (FA-BCIP) is a multiplex polymerase chain reaction that can detect 24 pathogens within 1 hour. We assessed whether results from FA-BCIP lead to changes in clinical management in a tertiary referral paediatric hospital. METHODS: We prospectively studied children having blood cultures taken at our tertiary children's hospital. Blood cultures were monitored and organisms identified using standard methods. FA-BCIP was performed when growth was initially detected in first positive blood cultures per episode, between January 1 and June 30, 2014. Assessment of whether the FA-BCIP result altered clinical management was made, specifically focused on antimicrobial stewardship and length of stay. RESULTS: FA-BCIP was done on 117 positive blood cultures; 74 (63%) grew clinically significant organisms, 43 (37%) grew contaminants. FA-BCIP results were judged to alter clinical management in 63 of the 117 episodes (54%). Antimicrobials were started/altered in 23 (19%) episodes and de-escalated/withheld/stopped in 29 (25%) episodes. Ten children were discharged from hospital earlier, which saved a cumulative total of 14 bed days. CONCLUSIONS: Rapid identification of microorganisms in pediatric blood cultures by FA-BCIP, led to changes in clinical management for half of the episodes. This improved antimicrobial stewardship and allowed early discharge from hospital for 10% of children. Future studies should focus on how best to use this technology in a cost-effective manner.

Inherent properties of adenylosuccinate lyase could explain S-Ado/SAICAr ratio due to homozygous R426H and R303C mutations.

Adenylosuccinate lyase (ADSL) is a homotetrameric enzyme involved in the de novo purine biosynthesis pathway and purine nucleotide cycle. Missense mutations in the protein lead to ADSL deficiency, an inborn error of purine metabolism characterized by neurological and physiological symptoms. ADSL deficiency is biochemically diagnosed by elevated levels of succinylaminoimidazolecarboxamide riboside (SAICAr) and succinyladenosine (S-Ado), the dephosphorylated derivatives of the substrates. S-Ado/SAICAr ratios have been associated with three phenotypic groups. Different hypotheses to explain these ratios have been proposed. Recent studies have focused on measuring activity on the substrates independently. However, it is important to examine mixtures of the substrates to determine if mutations affect enzyme activity on both substrates similarly in these conditions. The two substrates may experience an indirect communication due to being acted upon by the same enzyme, altering their activities from the non-competitive case. In this study, we investigate this hidden coupling between the two substrates. We chose two mutations that represent extremes of the phenotype, R426H and R303C. We describe a novel electrochemical-detection method of measuring the kinetic activity of ADSL in solution with its two substrates at varying concentration ratios. Furthermore, we develop an enzyme kinetic model to predict substrate activity from a given ratio of substrate concentrations. Our findings indicate a non-linear dependence of the activities on the substrate ratios due to competitive binding, distinct differences in the behaviors of the different mutations, and S-Ado/SAICAr ratios in patients could be explained by inherent properties of the mutant enzyme.

Structural and biochemical characterization of human adenylosuccinate lyase (ADSL) and the R303C ADSL deficiency-associated mutation.

Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive disorder, which causes a defect in purine metabolism resulting in neurological and physiological symptoms. ADSL executes two nonsequential steps in the de novo synthesis of AMP: the conversion of phosphoribosylsuccinyl-aminoimidazole carboxamide (SAICAR) to phosphoribosylaminoimidazole carboxamide, which occurs in the de novo synthesis of IMP, and the conversion of adenylosuccinate to AMP, which occurs in the de novo synthesis of AMP and also in the purine nucleotide cycle, using the same active site. Mutation of ADSL's arginine 303 to a cysteine is known to lead to ADSL deficiency. Interestingly, unlike other mutations leading to ADSL deficiency, the R303C mutation has been suggested to more significantly affect the enzyme's ability to catalyze the conversion of succinyladenosine monophosphate than that of SAICAR to their respective products. To better understand the causation of disease due to the R303C mutation, as well as to gain insights into why the R303C mutation potentially has a disproportional decrease in activity toward its substrates, the wild type (WT) and the R303C mutant of ADSL were investigated enzymatically and thermodynamically. Additionally, the X-ray structures of ADSL in its apo form as well as with the R303C mutation were elucidated, providing insight into ADSL's cooperativity. By utilizing this information, a model for the interaction between ADSL and SAICAR is proposed.