Diagnostic Validation of the Updated Pediatric Sepsis Biomarker Risk II for Acute Kidney Injury Prediction Model in Pediatric Septic Shock.

OBJECTIVES: We previously derived the updated Pediatric Sepsis Biomarker Risk for Acute Kidney Injury (PERSEVERE-II AKI) prediction model, which had robust diagnostic test characteristics for severe AKI on day 3 (D3 severe AKI) of septic shock. We now sought to validate this model in an independent cohort of children to the one in which the model was developed. DESIGN: A secondary analysis of a multicenter, prospective, observational study carried out from January 2019 to December 2022. SETTING: Ten PICUs in the United States. PATIENTS: Children with septic shock 1 week to 18 years old admitted to the PICU. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Seventy-nine of 363 patients (22%) had D3 severe AKI, defined as Kidney Disease Improving Global Outcomes stage 2 or higher. Patients were assigned a probability of D3 severe AKI using the PERSEVERE-II AKI model. The model predicted D3 severe AKI with an area under the receiver operating characteristic curve of 0.89 (95% CI, 0.85-0.93), sensitivity of 77% (95% CI, 66-86%), specificity of 88% (95% CI, 84-92%), positive predictive value of 65% (95% CI, 54-74%), and negative predictive value of 93% (95% CI, 89-96%). These data represent an increase in post-test probability of D3 severe AKI with a positive test from 22% to 65%, and a prevalence threshold of 28%. On multivariable regression, the PERSEVERE-II AKI prediction model demonstrated greater adjusted odds ratio (aOR) for D3 severe AKI (aOR, 11.2; 95% CI, 4.9-25.3) and lesser aOR for failure of D3 renal recovery from early AKI (aOR, 0.31; 95% CI, 0.13-0.69). CONCLUSIONS: The PERSEVERE-II AKI model demonstrates consistently robust performance for prediction of new or persistent D3 severe AKI in children with septic shock. A major limitation is that actual D3 severe AKI prevalence is below the prevalence threshold for the test, and thus future work should focus on evaluating use in enriched populations.

Prognostic and predictive value of endothelial dysfunction biomarkers in sepsis-associated acute kidney injury: risk-stratified analysis from a prospective observational cohort of pediatric septic shock.

BACKGROUND: Sepsis-associated acute kidney injury (SA-AKI) is associated with high morbidity, with no current therapies available beyond continuous renal replacement therapy (CRRT). Systemic inflammation and endothelial dysfunction are key drivers of SA-AKI. We sought to measure differences between endothelial dysfunction markers among children with and without SA-AKI, test whether this association varied across inflammatory biomarker-based risk strata, and develop prediction models to identify those at highest risk of SA-AKI. METHODS: Secondary analyses of prospective observational cohort of pediatric septic shock. Primary outcome of interest was the presence of ≥ Stage II KDIGO SA-AKI on day 3 based on serum creatinine (D3 SA-AKI SCr). Biomarkers including those prospectively validated to predict pediatric sepsis mortality (PERSEVERE-II) were measured in Day 1 (D1) serum. Multivariable regression was used to test the independent association between endothelial markers and D3 SA-AKI SCr. We conducted risk-stratified analyses and developed prediction models using Classification and Regression Tree (CART), to estimate risk of D3 SA-AKI among prespecified subgroups based on PERSEVERE-II risk. RESULTS: A total of 414 patients were included in the derivation cohort. Patients with D3 SA-AKI SCr had worse clinical outcomes including 28-day mortality and need for CRRT. Serum soluble thrombomodulin (sTM), Angiopoietin-2 (Angpt-2), and Tie-2 were independently associated with D3 SA-AKI SCr. Further, Tie-2 and Angpt-2/Tie-2 ratios were influenced by the interaction between D3 SA-AKI SCr and risk strata. Logistic regression demonstrated models predictive of D3 SA-AKI risk performed optimally among patients with high- or intermediate-PERSEVERE-II risk strata. A 6 terminal node CART model restricted to this subgroup of patients had an area under the receiver operating characteristic curve (AUROC) 0.90 and 0.77 upon tenfold cross-validation in the derivation cohort to distinguish those with and without D3 SA-AKI SCr and high specificity. The newly derived model performed modestly in a unique set of patients (n = 224), 84 of whom were deemed high- or intermediate-PERSEVERE-II risk, to distinguish those patients with high versus low risk of D3 SA-AKI SCr. CONCLUSIONS: Endothelial dysfunction biomarkers are independently associated with risk of severe SA-AKI. Pending validation, incorporation of endothelial biomarkers may facilitate prognostic and predictive enrichment for selection of therapeutics in future clinical trials among critically ill children.

Detrimental effects of PCSK9 loss-of-function in the pediatric host response to sepsis are mediated through independent influence on Angiopoietin-1.

BACKGROUND: Sepsis is associated with significant mortality. Yet, there are no efficacious therapies beyond antibiotics. PCSK9 loss-of-function (LOF) and inhibition, through enhanced low-density lipoprotein receptor (LDLR) mediated endotoxin clearance, holds promise as a potential therapeutic approach among adults. In contrast, we have previously demonstrated higher mortality in the juvenile host. Given the potential pleiotropic effects of PCSK9 on the endothelium, beyond canonical effects on serum lipoproteins, both of which may influence sepsis outcomes, we sought to test the influence of PCSK9 LOF genotype on endothelial dysfunction. METHODS: Secondary analyses of a prospective observational cohort of pediatric septic shock. Genetic variants of PCSK9 and LDLR genes, serum PCSK9, and lipoprotein concentrations were determined previously. Endothelial dysfunction markers were measured in day 1 serum. We conducted multivariable linear regression to test the influence of PCSK9 LOF genotype on endothelial markers, adjusted for age, complicated course, and low- and high-density lipoproteins (LDL and HDL). Causal mediation analyses to test impact of select endothelial markers on the association between PCSK9 LOF genotype and mortality. Juvenile Pcsk9 null and wildtype mice were subject to cecal slurry sepsis and endothelial markers were quantified. RESULTS: A total of 474 patients were included. PCSK9 LOF was associated with several markers of endothelial dysfunction, with strengthening of associations after exclusion of those homozygous for the rs688 LDLR variant that renders it insensitive to PCSK9. Serum PCSK9 was not correlated with endothelial dysfunction. PCSK9 LOF influenced concentrations of Angiopoietin-1 (Angpt-1) upon adjusting for potential confounders including lipoprotein concentrations, with false discovery adjusted p value of 0.042 and 0.013 for models that included LDL and HDL, respectively. Causal mediation analysis demonstrated that the effect of PCSK9 LOF on mortality was mediated by Angpt-1 (p = 0.0008). Murine data corroborated these results with lower Angpt-1 and higher soluble thrombomodulin among knockout mice with sepsis relative to the wildtype. CONCLUSIONS: We present genetic and biomarker association data that suggest a potential direct role of the PCSK9-LDLR pathway on Angpt-1 in the developing host with septic shock and warrant external validation. Further, mechanistic studies on the role of PCSK9-LDLR pathway on vascular homeostasis may lead to the development of pediatric-specific sepsis therapies.

Serum renin and prorenin concentrations predict severe persistent acute kidney injury and mortality in pediatric septic shock.

BACKGROUND: Studies in critically ill adults demonstrate associations between serum renin concentrations (a proposed surrogate for renin-angiotensin-aldosterone system dysregulation) and poor outcomes, but data in critically ill children are lacking. We assessed serum renin + prorenin concentrations in children with septic shock to determine their predictive ability for acute kidney injury (AKI) and mortality. METHODS: We conducted a secondary analysis of a multicenter observational study of children aged 1 week to 18 years admitted to 14 pediatric intensive care units (PICUs) with septic shock and residual serum available for renin + prorenin measurement. Primary outcomes were development of severe persistent AKI (≥ KDIGO stage 2 for ≥ 48 h) in the first week and 28-day mortality. RESULTS: Among 233 patients, day 1 median renin + prorenin concentration was 3436 pg/ml (IQR 1452-6567). Forty-two (18%) developed severe persistent AKI and 32 (14%) died. Day 1 serum renin + prorenin predicted severe persistent AKI with an AUROC of 0.75 (95% CI 0.66-0.84, p < 0.0001; optimal cutoff 6769 pg/ml) and mortality with an AUROC of 0.79 (95% CI 0.69-0.89, p < 0.0001; optimal cutoff 6521 pg/ml). Day 3/day 1 (D3:D1) renin + prorenin ratio had an AUROC of 0.73 (95% CI 0.63-0.84, p < 0.001) for mortality. On multivariable regression, day 1 renin + prorenin > optimal cutoff retained associations with severe persistent AKI (aOR 6.8, 95% CI 3.0-15.8, p < 0.001) and mortality (aOR 6.9, 95% CI 2.2-20.9, p < 0.001). Similarly, D3:D1 renin + prorenin > optimal cutoff was associated with mortality (aOR 7.6, 95% CI 2.5-23.4, p < 0.001). CONCLUSIONS: Children with septic shock have very elevated serum renin + prorenin concentrations on PICU admission, and these concentrations, as well as their trend over the first 72 h, predict severe persistent AKI and mortality. A higher resolution version of the Graphical abstract is available as Supplementary information.

Integrated PERSEVERE and endothelial biomarker risk model predicts death and persistent MODS in pediatric septic shock: a secondary analysis of a prospective observational study.

BACKGROUND: Multiple organ dysfunction syndrome (MODS) is a critical driver of sepsis morbidity and mortality in children. Early identification of those at risk of death and persistent organ dysfunctions is necessary to enrich patients for future trials of sepsis therapeutics. Here, we sought to integrate endothelial and PERSEVERE biomarkers to estimate the composite risk of death or organ dysfunctions on day 7 of septic shock. METHODS: We measured endothelial dysfunction markers from day 1 serum among those with existing PERSEVERE data. TreeNet® classification model was derived incorporating 22 clinical and biological variables to estimate risk. Based on relative variable importance, a simplified 6-biomarker model was developed thereafter. RESULTS: Among 502 patients, 49 patients died before day 7 and 124 patients had persistence of MODS on day 7 of septic shock. Area under the receiver operator characteristic curve (AUROC) for the newly derived PERSEVEREnce model to predict death or day 7 MODS was 0.93 (0.91-0.95) with a summary AUROC of 0.80 (0.76-0.84) upon tenfold cross-validation. The simplified model, based on IL-8, HSP70, ICAM-1, Angpt2/Tie2, Angpt2/Angpt1, and Thrombomodulin, performed similarly. Interaction between variables-ICAM-1 with IL-8 and Thrombomodulin with Angpt2/Angpt1-contributed to the models' predictive capabilities. Model performance varied when estimating risk of individual organ dysfunctions with AUROCS ranging from 0.91 to 0.97 and 0.68 to 0.89 in training and test sets, respectively. CONCLUSIONS: The newly derived PERSEVEREnce biomarker model reliably estimates risk of death or persistent organ dysfunctions on day 7 of septic shock. If validated, this tool can be used for prognostic enrichment in future pediatric trials of sepsis therapeutics.

PERSEVERE Biomarkers Predict Severe Acute Kidney Injury and Renal Recovery in Pediatric Septic Shock.

Rationale: Acute kidney injury (AKI), a common complication of sepsis, is associated with substantial morbidity and mortality and lacks definitive disease-modifying therapy. Early, reliable identification of at-risk patients is important for targeted implementation of renal protective measures. The updated Pediatric Sepsis Biomarker Risk Model (PERSEVERE-II) is a validated, multibiomarker prognostic enrichment strategy to estimate baseline mortality risk in pediatric septic shock.Objectives: To assess the association between PERSEVERE-II mortality probability and the development of severe, sepsis-associated AKI on Day 3 (D3 SA-AKI) in pediatric septic shock.Methods: We performed secondary analysis of a prospective observational study of children with septic shock in whom the PERSEVERE biomarkers were measured to assign a PERSEVERE-II baseline mortality risk.Measurements and Main Results: Among 379 patients, 65 (17%) developed severe D3 SA-AKI. The proportion of patients developing severe D3 SA-AKI increased directly with increasing PERSEVERE-II risk category, and increasing PERSEVERE-II mortality probability was independently associated with increased odds of severe D3 SA-AKI after adjustment for age and illness severity (odds ratio, 1.4; 95% confidence interval, 1.2-1.7; P < 0.001). Similar associations were found between increasing PERSEVERE-II mortality probability and the need for renal replacement therapy. Lower PERSEVERE-II mortality probability was independently associated with increased odds of renal recovery among patients with early AKI. A newly derived model incorporating the PERSEVERE biomarkers and Day 1 AKI status predicted severe D3 SA-AKI with an area under the received operating characteristic curve of 0.95 (95% confidence interval, 0.92-0.98).Conclusions: Among children with septic shock, the PERSEVERE biomarkers predict severe D3 SA-AKI and identify patients with early AKI who are likely to recover.

Proprotein Convertase Subtilisin/Kexin Type 9 Loss-of-Function Is Detrimental to the Juvenile Host With Septic Shock.

OBJECTIVES: Proprotein convertase subtilisin/kexin type 9 is a central regulator of lipid metabolism and has been implicated in regulating the host response to sepsis. Proprotein convertase subtilisin/kexin type 9 loss-of-function is associated with improved sepsis outcomes in the adult host through increased hepatic bacterial clearance. Thus, there is interest in leveraging proprotein convertase subtilisin/kexin type 9 inhibitors as a therapeutic strategy in adults with sepsis. We sought to validate this association in children with septic shock and in a juvenile murine model of sepsis. DESIGN: Prospectively enrolled cohort of children with septic shock; experimental mice. SETTING: Seventeen participating institutions; research laboratory. PATIENTS AND SUBJECTS: Five-hundred twenty-two children with septic shock; juvenile (14 d old) and adult (10-14 wk) mice with constitutive proprotein convertase subtilisin/kexin type 9 null and wildtype control mice (C57BL/6). INTERVENTIONS: Proprotein convertase subtilisin/kexin type 9 single-nucleotide polymorphisms, serum proprotein convertase subtilisin/kexin type 9, and lipid profiles in patients. Cecal slurry murine model of sepsis; survival studies in juvenile and adult mice, assessment of lipoprotein fractions, bacterial burden, and inflammation in juvenile mice. MEASUREMENTS AND MAIN RESULTS: PCSK9 loss-of-function genetic variants were independently associated with increased odds of complicated course and mortality in children with septic shock. PCSK9, low-density lipoprotein, and high-density lipoprotein concentrations were lower among patients with complicated course relative to those without. PCSK9 concentrations negatively correlated with proinflammatory cytokine interleukin-8. Proprotein convertase subtilisin/kexin type 9 loss-of-function decreased survival in juvenile mice, but increased survival in adult mice with sepsis. PCSK9 loss-of-function resulted in low lipoproteins and decreased hepatic bacterial burden in juvenile mice. CONCLUSIONS: In contrast to the adult host, proprotein convertase subtilisin/kexin type 9 loss-of-function is detrimental to the juvenile host with septic shock. PCSK9 loss-of-function, in the context of low lipoproteins, may result in reduced hepatic bacterial clearance in the juvenile host with septic shock. Our data indicate that children should be excluded in sepsis clinical trials involving proprotein convertase subtilisin/kexin type 9 inhibitors.

Endotype Transitions During the Acute Phase of Pediatric Septic Shock Reflect Changing Risk and Treatment Response.

OBJECTIVE: We previously identified septic shock endotypes A and B based on 100 genes reflecting adaptive immunity and glucocorticoid receptor signaling. The endotypes differ with respect to outcome and corticosteroid responsiveness. We determined whether endotypes change during the initial 3 days of illness, and whether changes are associated with outcomes. DESIGN: Observational cohort study including existing and newly enrolled participants. SETTING: Multiple PICUs. PATIENTS: Children with septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We measured the 100 endotyping genes at day 1 and day 3 of illness in 375 patients. We determined if endotype assignment changes over time, and whether changing endotype is associated with corticosteroid response and outcomes. We used multivariable logistic regression to adjust for illness severity, age, and comorbidity burden. Among the 132 subjects assigned to endotype A on day 1, 56 (42%) transitioned to endotype B by day 3. Among 243 subjects assigned to endotype B on day 1, 77 (32%) transitioned to endotype A by day 3. Assignment to endotype A on day 1 was associated with increased odds of mortality. This risk was modified by the subsequent day 3 endotype assignment. Corticosteroids were associated with increased risk of mortality among subjects who persisted as endotype A. CONCLUSIONS: A substantial proportion of children with septic shock transition endotypes during the acute phase of illness. The risk of poor outcome and the response to corticosteroids change with changes in endotype assignment. Patients persisting as endotype A are at highest risk of poor outcomes.

Hyperchloremia Is Associated With Complicated Course and Mortality in Pediatric Patients With Septic Shock.

OBJECTIVE: Hyperchloremia is associated with poor outcome among critically ill adults, but it is unknown if a similar association exists among critically ill children. We determined if hyperchloremia is associated with poor outcomes in children with septic shock. DESIGN: Retrospective analysis of a pediatric septic shock database. SETTING: Twenty-nine PICUs in the United States. PATIENTS: Eight hundred ninety children 10 years and younger with septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We considered the minimum, maximum, and mean chloride values during the initial 7 days of septic shock for each study subject as separate hyperchloremia variables. Within each category, we considered hyperchloremia as a dichotomous variable defined as a serum concentration greater than or equal to 110 mmol/L. We used multivariable logistic regression to determine the association between the hyperchloremia variables and outcome, adjusted for illness severity. We considered all cause 28-day mortality and complicated course as the primary outcome variables. Complicated course was defined as mortality by 28 days or persistence of greater than or equal to two organ failures at day 7 of septic shock. Secondarily, we conducted a stratified analysis using a biomarker-based mortality risk stratification tool. There were 226 patients (25%) with a complicated course and 93 mortalities (10%). Seventy patients had a minimum chloride greater than or equal to 110 mmol/L, 179 had a mean chloride greater than or equal to 110 mmol/L, and 514 had a maximum chloride greater than or equal to 110 mmol/L. A minimum chloride greater than or equal to 110 mmol/L was associated with increased odds of complicated course (odds ratio, 1.9; 95% CI, 1.1-3.2; p = 0.023) and mortality (odds ratio, 3.7; 95% CI, 2.0-6.8; p < 0.001). A mean chloride greater than or equal to 110 mmol/L was also associated with increased odds of mortality (odds ratio, 2.1; 95% CI, 1.3-3.5; p = 0.002). The secondary analysis yielded similar results. CONCLUSION: Hyperchloremia is independently associated with poor outcomes among children with septic shock.

Improved Risk Stratification in Pediatric Septic Shock Using Both Protein and mRNA Biomarkers. PERSEVERE-XP.

RATIONALE: We previously derived and validated the Pediatric Sepsis Biomarker Risk Model (PERSEVERE) to estimate baseline mortality risk in children with septic shock. The PERSEVERE biomarkers are serum proteins selected from among the proteins directly related to 80 mortality risk assessment genes. The initial approach to selecting the PERSEVERE biomarkers left 68 genes unconsidered. OBJECTIVES: To determine if the 68 previously unconsidered genes can improve upon the performance of PERSEVERE and to provide biological information regarding the pathophysiology of septic shock. METHODS: We reduced the number of variables by determining the biological linkage of the 68 previously unconsidered genes. The genes identified through variable reduction were combined with the PERSEVERE-based mortality probability to derive a risk stratification model for 28-day mortality using classification and regression tree methodology (n = 307). The derived tree, PERSEVERE-XP, was then tested in a separate cohort (n = 77). MEASUREMENTS AND MAIN RESULTS: Variable reduction revealed a network consisting of 18 mortality risk assessment genes related to tumor protein 53 (TP53). In the derivation cohort, PERSEVERE-XP had an area under the receiver operating characteristic curve (AUC) of 0.90 (95% confidence interval, 0.85-0.95) for differentiating between survivors and nonsurvivors. In the test cohort, the AUC was 0.96 (95% confidence interval, 0.91-1.0). The AUC of PERSEVERE-XP was superior to that of PERSEVERE. CONCLUSIONS: PERSEVERE-XP combines protein and mRNA biomarkers to provide mortality risk stratification with possible clinical utility. PERSEVERE-XP significantly improves on PERSEVERE and suggests a role for TP53-related cellular division, repair, and metabolism in the pathophysiology of septic shock.

Multiple Organ Dysfunction in Children Mechanically Ventilated for Acute Respiratory Failure.

OBJECTIVES: The impact of extrapulmonary organ dysfunction, independent from sepsis and lung injury severity, on outcomes in pediatric acute respiratory failure is unclear. We sought to determine the frequency, timing, and risk factors for extrapulmonary organ dysfunction and the independent association of multiple organ dysfunction syndrome with outcomes in pediatric acute respiratory failure. DESIGN: Secondary observational analysis of the Randomized Evaluation of Sedation Titration for Respiratory Failure cluster-randomized prospective clinical trial conducted between 2009 and 2013. SETTING: Thirty-one academic PICUs in the United States. PATIENTS: Two thousand four hundred forty-nine children mechanically ventilated for acute respiratory failure enrolled in Randomized Evaluation of Sedation Titration for Respiratory Failure. MEASUREMENTS AND MAIN RESULTS: Organ dysfunction was defined using criteria published for pediatric sepsis. Multiple organ dysfunction syndrome was defined as respiratory dysfunction one or more extrapulmonary organ dysfunctions. We used multivariable logistic regression to identify risk factors for multiple organ dysfunction syndrome, and logistic or proportional hazards regression to compare clinical outcomes. All analyses accounted for PICU as a cluster variable. Overall, 73% exhibited extrapulmonary organ dysfunction, including 1,547 (63%) with concurrent multiple organ dysfunction syndrome defined by onset on day 0/1 and 244 (10%) with new multiple organ dysfunction syndrome with onset on day 2 or later. Most patients (93%) with indirect lung injury from sepsis presented with concurrent multiple organ dysfunction syndrome, whereas patients with direct lung injury had both concurrent (56%) and new (12%) multiple organ dysfunction syndrome. Risk factors for concurrent multiple organ dysfunction syndrome included older age, illness severity, sepsis, cancer, and moderate/severe lung injury. Risk factors for new multiple organ dysfunction syndrome were moderate/severe lung injury and neuromuscular blockade. Both concurrent and new multiple organ dysfunction syndrome were associated with 90-day in-hospital mortality (concurrent: adjusted odds ratio, 6.54; 95% CI, 3.00-14.25 and new: adjusted odds ratio, 3.21; 95% CI, 1.48-6.93) after adjusting for sepsis, moderate/severe lung injury, and other baseline characteristics. CONCLUSIONS: Extrapulmonary organ dysfunction was common, generally occurred concurrent with respiratory dysfunction (especially in sepsis), and was a major risk factor for mortality in pediatric acute respiratory failure.

Glucocorticoid Receptor Polymorphisms and Outcomes in Pediatric Septic Shock.

OBJECTIVE: Polymorphisms of the glucocorticoid receptor gene are associated with outcome and corticosteroid responsiveness among patients with inflammatory disorders. We conducted a candidate gene association study to test the hypothesis that these polymorphisms are associated with outcome and corticosteroid responsiveness among children with septic shock. DESIGN: We genotyped 482 children with septic shock for the presence of two glucocorticoid receptor polymorphisms (rs56149945 and rs41423247) associated with increased sensitivity and one glucocorticoid receptor polymorphism (rs6198) associated with decreased sensitivity to corticosteroids. The primary outcome variable was complicated course, defined as 28-day mortality or the persistence of two or more organ failures 7 days after a septic shock diagnosis. We used logistic regression to test for an association between corticosteroid exposure and outcome, within genotype group, and adjusted for illness severity. SETTING: Multiple PICUs in the United States. INTERVENTIONS: Standard care. MEASUREMENTS AND MAIN RESULTS: There were no differences in outcome when comparing the various genotype groups. Among patients homozygous for the wild-type glucocorticoid receptor allele, corticosteroids were independently associated with increased odds of complicated course (odds ratio, 2.30; 95% CI, 1.01-5.21; p = 0.047). CONCLUSIONS: Based on these glucocorticoid receptor polymorphisms, we could not detect a beneficial effect of corticosteroids among any genotype group. Among children homozygous for the wild-type allele, corticosteroids were independently associated with increased odds of poor outcome.

Combining Prognostic and Predictive Enrichment Strategies to Identify Children With Septic Shock Responsive to Corticosteroids.

OBJECTIVES: Prognostic and predictive enrichment strategies are fundamental tools of precision medicine. Identifying children with septic shock who may benefit from corticosteroids remains a challenge. We combined prognostic and predictive strategies to identify a pediatric septic shock subgroup responsive to corticosteroids. DESIGN: We conducted a secondary analysis of 288 previously published pediatric subjects with septic shock. For prognostic enrichment, each study subject was assigned a baseline mortality probability using the pediatric sepsis biomarker risk model. For predictive enrichment, each study subject was allocated to one of two septic shock endotypes, based on a 100-gene signature reflecting adaptive immunity and glucocorticoid receptor signaling. The primary study endpoint was complicated course, defined as the persistence of two or more organ failures at day 7 of septic shock or 28-day mortality. We used logistic regression to test for an association between corticosteroids and complicated course within endotype. MEASUREMENTS AND MAIN RESULTS: Among endotype B subjects at intermediate to high pediatric sepsis biomarker risk model-based risk of mortality, corticosteroids were independently associated with more than a 10-fold reduction in the risk of a complicated course (relative risk, 0.09; 95% CI, 0.01-0.54; p = 0.007). CONCLUSIONS: A combination of prognostic and predictive strategies based on serum protein and messenger RNA biomarkers can identify a subgroup of children with septic shock who may be more likely to benefit from corticosteroids. Prospective validation of these strategies and the existence of this subgroup are warranted.

Pediatric Sepsis Biomarker Risk Model-II: Redefining the Pediatric Sepsis Biomarker Risk Model With Septic Shock Phenotype.

OBJECTIVE: The Pediatric Sepsis Biomarker Risk Model (PERSEVERE), a pediatric sepsis risk model, uses biomarkers to estimate baseline mortality risk for pediatric septic shock. It is unknown how PERSEVERE performs within distinct septic shock phenotypes. We tested PERSEVERE in children with septic shock and thrombocytopenia-associated multiple organ failure (TAMOF), and in those without new onset thrombocytopenia but with multiple organ failure (MOF). DESIGN: PERSEVERE-based mortality risk was generated for each study subject (n = 660). A priori, we determined that if PERSEVERE did not perform well in both the TAMOF and the MOF cohorts, we would revise PERSEVERE to incorporate admission platelet counts. SETTING: Multiple PICUs in the United States. INTERVENTIONS: Standard care. MEASUREMENTS AND MAIN RESULTS: PERSEVERE performed well in the TAMOF cohort (areas under the receiver operating characteristic curves [AUC], 0.84 [95% CI, 0.77-0.90]), but less well in the MOF cohort (AUC, 0.71 [0.61-0.80]). PERSEVERE was revised using 424 subjects previously reported in the derivation phase. PERSEVERE-II had an AUC of 0.89 (0.85-0.93) and performed equally well across TAMOF and MOF cohorts. PERSEVERE-II performed well when tested in 236 newly enrolled subjects. Sample size calculations for a clinical trial testing the efficacy of plasma exchange for children with septic shock and TAMOF indicated PERSEVERE-II-based stratification could substantially reduce the number of patients necessary, when compared with no stratification. CONCLUSIONS: Testing PERSEVERE in the context of septic shock phenotypes prompted a revision incorporating platelet count. PERSEVERE-II performs well upon testing, independent of TAMOF or MOF status. PERSEVERE-II could potentially serve as a prognostic enrichment tool.

Developing a clinically feasible personalized medicine approach to pediatric septic shock.

RATIONALE: Using microarray data, we previously identified gene expression-based subclasses of septic shock with important phenotypic differences. The subclass-defining genes correspond to adaptive immunity and glucocorticoid receptor signaling. Identifying the subclasses in real time has theranostic implications, given the potential for immune-enhancing therapies and controversies surrounding adjunctive corticosteroids for septic shock. OBJECTIVES: To develop and validate a real-time subclassification method for septic shock. METHODS: Gene expression data for the 100 subclass-defining genes were generated using a multiplex messenger RNA quantification platform (NanoString nCounter) and visualized using gene expression mosaics. Study subjects (n = 168) were allocated to the subclasses using computer-assisted image analysis and microarray-based reference mosaics. A gene expression score was calculated to reduce the gene expression patterns to a single metric. The method was tested prospectively in a separate cohort (n = 132). MEASUREMENTS AND MAIN RESULTS: The NanoString-based data reproduced two septic shock subclasses. As previously, one subclass had decreased expression of the subclass-defining genes. The gene expression score identified this subclass with an area under the curve of 0.98 (95% confidence interval [CI95] = 0.96-0.99). Prospective testing of the subclassification method corroborated these findings. Allocation to this subclass was independently associated with mortality (odds ratio = 2.7; CI95 = 1.2-6.0; P = 0.016), and adjunctive corticosteroids prescribed at physician discretion were independently associated with mortality in this subclass (odds ratio = 4.1; CI95 = 1.4-12.0; P = 0.011). CONCLUSIONS: We developed and tested a gene expression-based classification method for pediatric septic shock that meets the time constraints of the critical care environment, and can potentially inform therapeutic decisions.

A Multibiomarker-Based Model for Estimating the Risk of Septic Acute Kidney Injury.

OBJECTIVE: The development of acute kidney injury in patients with sepsis is associated with worse outcomes. Identifying those at risk for septic acute kidney injury could help to inform clinical decision making. We derived and tested a multibiomarker-based model to estimate the risk of septic acute kidney injury in children with septic shock. DESIGN: Candidate serum protein septic acute kidney injury biomarkers were identified from previous transcriptomic studies. Model derivation involved measuring these biomarkers in serum samples from 241 subjects with septic shock obtained during the first 24 hours of admission and then using a Classification and Regression Tree approach to estimate the probability of septic acute kidney injury 3 days after the onset of septic shock, defined as at least two-fold increase from baseline serum creatinine. The model was then tested in a separate cohort of 200 subjects. SETTING: Multiple PICUs in the United States. INTERVENTIONS: None other than standard care. MEASUREMENTS AND MAIN RESULTS: The decision tree included a first-level decision node based on day 1 septic acute kidney injury status and five subsequent biomarker-based decision nodes. The area under the curve for the tree was 0.95 (CI95, 0.91-0.99), with a sensitivity of 93% and a specificity of 88%. The tree was superior to day 1 septic acute kidney injury status alone for estimating day 3 septic acute kidney injury risk. In the test cohort, the tree had an area under the curve of 0.83 (0.72-0.95), with a sensitivity of 85% and a specificity of 77% and was also superior to day 1 septic acute kidney injury status alone for estimating day 3 septic acute kidney injury risk. CONCLUSIONS: We have derived and tested a model to estimate the risk of septic acute kidney injury on day 3 of septic shock using a novel panel of biomarkers. The model had very good performance in a test cohort and has test characteristics supporting clinical utility and further prospective evaluation.

Differential expression of the Nrf2-linked genes in pediatric septic shock.

INTRODUCTION: Experimental data from animal models of sepsis support a role for a transcription factor, nuclear erythroid-related factor 2 p45-related factor 2 (Nrf2), as a master regulator of antioxidant and detoxifying genes and intermediary metabolism during stress. Prior analysis of a pediatric septic shock transcriptomic database showed that the Nrf2 response is a top 5 upregulated signaling pathway in early pediatric septic shock. METHODS: We conducted a focused analysis of 267 Nrf2-linked genes using a multicenter, genome-wide expression database of 180 children with septic shock 10 years of age or younger and 53 healthy controls. The analysis involved RNA isolated from whole blood within 24 h of pediatric intensive care unit admission for septic shock and a false discovery rate of 5 %. We compared differentially expressed genes from (1) patients with septic shock and healthy controls and (2) across validated gene expression-based subclasses of pediatric septic shock (endotypes A and B) using several bioinformatic methods. RESULTS: We found upregulation of 123 Nrf2-linked genes in children with septic shock. The top gene network represented by these genes contained primarily enzymes with oxidoreductase activity involved in cellular lipid metabolism that were highly connected to the peroxisome proliferator activated receptor and the retinoic acid receptor families. Endotype A, which had higher organ failure burden and mortality, exhibited a greater downregulation of Nrf2-linked genes than endotype B, with 92 genes differentially regulated between endotypes. CONCLUSIONS: Our findings indicate that Nrf2-linked genes may contribute to alterations in oxidative signaling and intermediary metabolism in pediatric septic shock.

Corticosteroids are associated with repression of adaptive immunity gene programs in pediatric septic shock.

RATIONALE: Corticosteroids are prescribed commonly for patients with septic shock, but their use remains controversial and concerns remain regarding side effects. OBJECTIVES: To determine the effect of adjunctive corticosteroids on the genomic response of pediatric septic shock. METHODS: We retrospectively analyzed an existing transcriptomic database of pediatric septic shock. Subjects receiving any formulation of systemic corticosteroids at the time of blood draw for microarray analysis were classified in the septic shock corticosteroid group. We compared normal control subjects (n = 52), a septic shock no corticosteroid group (n = 110), and a septic shock corticosteroid group (n = 70) using analysis of variance. Genes differentially regulated between the no corticosteroid group and the corticosteroid group were analyzed using Ingenuity Pathway Analysis. MEASUREMENTS AND MAIN RESULTS: The two study groups did not differ with respect to illness severity, organ failure burden, mortality, or mortality risk. There were 319 gene probes differentially regulated between the no corticosteroid group and the corticosteroid group. These genes corresponded predominately to adaptive immunity-related signaling pathways, and were down-regulated relative to control subjects. Notably, the degree of down-regulation was significantly greater in the corticosteroid group, compared with the no corticosteroid group. A similar pattern was observed for genes corresponding to the glucocorticoid receptor signaling pathway. CONCLUSIONS: Administration of corticosteroids in pediatric septic shock is associated with additional repression of genes corresponding to adaptive immunity. These data should be taken into account when considering the benefit to risk ratio of adjunctive corticosteroids for septic shock.

Post-ICU admission fluid balance and pediatric septic shock outcomes: a risk-stratified analysis.

OBJECTIVE: Observed associations between fluid balance and septic shock outcomes are likely confounded by initial mortality risk. We conducted a risk-stratified analysis of the association between post-ICU admission fluid balance and pediatric septic shock outcomes. DESIGN: Retrospective analysis of an ongoing multicenter pediatric septic shock clinical and biological database. SETTING: Seventeen PICUs in the United States. PATIENTS: Three hundred and seventeen children with septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We stratified subjects into three mortality risk categories (low, intermediate, and high) using a validated biomarker-based stratification tool. Within each category, we assessed three fluid balance variables: total fluid intake/kg/d during the first 24 hours, percent positive fluid balance during the first 24 hours, and cumulative percent positive fluid balance up to 7 days. We used logistic regression to estimate the effect of fluid balance on the odds of 28-day mortality, and on complicated course, which we defined as either death within 28 days or persistence of two or more organ failures at 7 days. There were 40 deaths, and 91 subjects had a complicated course. Increased cumulative percent positive fluid balance was associated with mortality in the low-risk cohort (n = 204; odds ratio, 1.035; 95% CI, 1.004-1.066) but not in the intermediate- and high-risk cohorts. No other associations with mortality were observed. Fluid intake, percent positive fluid balance in the first 24 hours, and cumulative percent positive fluid balance were all associated with increased odds of a complicated course in the low-risk cohort but not in the intermediate- and high-risk cohorts. CONCLUSIONS: When stratified for mortality risk, increased fluid intake and positive fluid balance after ICU admission are associated with worse outcomes in pediatric septic shock patients with a low initial mortality risk but not in patients at moderate or high mortality risk.

Differential expression of the nuclear-encoded mitochondrial transcriptome in pediatric septic shock.

INTRODUCTION: Increasing evidence supports a role for mitochondrial dysfunction in organ injury and immune dysregulation in sepsis. Although differential expression of mitochondrial genes in blood cells has been reported for several diseases in which bioenergetic failure is a postulated mechanism, there are no data about the blood cell mitochondrial transcriptome in pediatric sepsis. METHODS: We conducted a focused analysis using a multicenter genome-wide expression database of 180 children ≤ 10 years of age with septic shock and 53 healthy controls. Using total RNA isolated from whole blood within 24 hours of PICU admission for septic shock, we evaluated 296 nuclear-encoded mitochondrial genes using a false discovery rate of 1%. A series of bioinformatic approaches were applied to compare differentially expressed genes across previously validated gene expression-based subclasses (groups A, B, and C) of pediatric septic shock. RESULTS: In total, 118 genes were differentially regulated in subjects with septic shock compared to healthy controls, including 48 genes that were upregulated and 70 that were downregulated. The top scoring canonical pathway was oxidative phosphorylation, with general downregulation of the 51 genes corresponding to the electron transport system (ETS). The top two gene networks were composed primarily of mitochondrial ribosomal proteins highly connected to ETS complex I, and genes encoding for ETS complexes I, II, and IV that were highly connected to the peroxisome proliferator activated receptor (PPAR) family. There were 162 mitochondrial genes differentially regulated between groups A, B, and C. Group A, which had the highest maximum number of organ failures and mortality, exhibited a greater downregulation of mitochondrial genes compared to groups B and C. CONCLUSIONS: Based on a focused analysis of a pediatric septic shock transcriptomic database, nuclear-encoded mitochondrial genes were differentially regulated early in pediatric septic shock compared to healthy controls, as well as across genotypic and phenotypic distinct pediatric septic shock subclasses. The nuclear genome may be an important mechanism contributing to alterations in mitochondrial bioenergetic function and outcomes in pediatric sepsis.