Project description:Background: Systemic inflammation is a whole body reaction that can have an infection-positive (i.e. sepsis) or infection-negative origin. It is important to distinguish between septic and non-septic presentations early and reliably, because this has significant therapeutic implications for critically ill patients. We hypothesized that a molecular classifier based on a small number of RNAs expressed in peripheral blood could be discovered that would: 1) determine which patients with systemic inflammation had sepsis; 2) be robust across independent patient cohorts; 3) be insensitive to disease severity; and 4) provide diagnostic utility. The overall goal of this study was to identify and validate such a molecular classifier. Methods and Findings: We conducted an observational, non-interventional study of adult patients recruited from tertiary intensive care units (ICU). Biomarker discovery was conducted with an Australian cohort (n = 105) consisting of sepsis patients and post -surgical patients with infection-negative systemic inflammation. Using this cohort, a four-gene classifier consisting of a combination of CEACAM4, LAMP1, PLA2G7 and PLAC8 RNA biomarkers was identified. This classifier, designated SeptiCyteÂ® Lab, was externally validated using RT-qPCR and receiver operating characteristic (ROC) curve analysis in five cohorts (n = 345) from the Netherlands. Cohort 1 (n=59) consisted of unambiguous septic cases and infection-negative systemic inflammation controls; SeptiCyteÂ® Lab gave an area under curve (AUC) of 0.96 (95% CI: 0.91-1.00). ROC analysis of a more heterogeneous group of patients (Cohorts 2-5; 249 patients after excluding 37 patients with infection likelihood possible) gave an AUC of 0.89 (95% CI: 0.85-0.93). Disease severity, as measured by Sequential Organ Failure Assessment (SOFA) score or the Acute Physiology and Chronic Health Evaluation (APACHE) IV score, was not a significant confounding variable. The diagnostic utility o f SeptiCyteÂ® Lab was evaluated by comparison to various clinical and laboratory parameters that would be available to a clinician within 24 hours of ICU admission. SeptiCyteÂ® Lab was significantly better at differentiating sepsis from infection-negative systemic inflammation than all tested parameters, both singly and in various logistic combinations. SeptiCyteÂ® Lab more than halved the diagnostic error rate compared to PCT in all tested cohorts or cohort combinations. Conclusions: SeptiCyteÂ® Lab is a rapid molecular assay that may be clinically useful in the management of ICU patients with systemic inflammation. SIRS and Sepsis ICU patients, admission samples Retrospective, mutli-site sutdy using retrospective physician adjudication as a comparator

Project description:BACKGROUND: Despite best supportive intensive care, the development of severe sepsis and septic shock after trauma is still associated with a high mortality rate in intensive care units (ICU). Incomplete knowledge of the pathophysiological and biochemical mechanisms that lead to perturbations of the host system is a major cause for this clinical entity. This lack of knowledge is mirrored in a delay in both diagnosis of sepsis and stratification of patients at risk. The current diagnostic and classification methods do not provide an early and reliable evidence for the development of sepsis and consequently lead to uncertainty in the classification and a delay in the initiation of an adequate therapy. Here, we present a prospective study performed with a well-defined patient cohort suffering from severe multiple trauma (n=85), in which we aimed to uncover the biological reasons why patients with multiple trauma can have dramatically different outcomes after suffering similar traumatic insults. METHODS: We used peripheral whole blood obtained with the PaxGene system at the time of admission to the ICU, i.e., 12 hours after trauma, for transcriptome analysis and monitored clinically the sequence of events from the admission to the ICU, to the very onset of sepsis and finally to the full development of multiple organ dysfunction syndrome (MODS). Study protocols and standard operating procedures (SOPs) regarding ethics, patient recruitment, logistics in sample acquisition and storage, microarray experiments, data analysis and data management have been developed and provided for a high feasibility and reproducibility of the investigations. FINDINGS: The transcriptome analysis from peripheral blood revealed a strong host response upon severe injury and demonstrated the suitability of whole blood in the PaxGene system as a surrogate marker in clinical gene expression studies. It was evident that patients who developed sepsis in the course of the disease (3-5 days after admission) showed an imbalance in the TH1/TH2 response towards a pronounced TH2 response, soon after trauma. The imbalanced TH1/TH2 shift along with inactivation of T cells, neutrophils, monocytes and macrophages might be critical factors for the initiation of a septic program seen in these patients 12 hours after trauma. INTERPRETATION: First, in this study, we provide protocols for logistics and infrastructure that can be applied generally to successfully integrate gene expression analysis studies in a clinical routine and demonstrate that by following these protocols, dedicated and reproducible results can be obtained. Second, we showed that peripheral whole blood is a suitable surrogate marker for the host response as it contains lots of “information” and is easily accessible. Third, we identified mechanisms involved in the perturbations of the immune system that lead to sepsis. Keywords: Prospective study 159 consecutive traumatized patients were included in the study upon admission to the intensive care unit (ICU), fulfilling all of the following inclusion criteria: severe injuries to at least two body regions or three major fractures, between 18 and 65 years of age, an estimated Injury Severity Score (ISS) of ?15 points after thorough assessment of injuries, <12 hours between the occurrence of the accident and the time of admission to the ICU, and at least greater than 3 days of survival. None of the patients underwent neuro- or cardiac surgery. We excluded patients with severe intracranial head injuries, coagulation abnormalities known at the day of admission to the ICU, acute renal failure, liver failure, malignant disease, or hemofiltration in the patient`s history. The Patients were divided into two groups depending on their posttraumatic course: Patients not complicatd by sepsis (Group: N) and Patients complicated by sepsis (Group: S). Using the CodeLink UniSet Human 10 K Bioarrays (GE Healthcare, Freiburg, Germany), we searched for early differences in the transcriptome of these two groups. Only whole blood samples drawn upon admission to the ICU were used for this investigation. Microarray results were validated by quantification of mRNA by TaqMan® technology. Each patient sample was hybridized in duplicate or triplicate on microarrays (label-extract technical replicate). A total of 72 arrays (36 group N, 36 group S) were subjected to microarray quality analysis. Of these, 2 arrays (0 group N, 2 group S) were excluded from the data set due to quality reasons and a final set of 70 arrays (36 group N and 34 group S) were subjected to microarray analysis. Since the eliminated 2 arrays belonged to patients with three replicates, they were treated as two replicates in the further analysis. The arrays were designated N_xx_yy_z or S_xx_yy_z, with x for the patient-ID (1,2,3,4..) and z for the technical replicate number (1,2 or 3).

Project description:Background: Systemic inflammation is a whole body reaction that can have an infection-positive (i.e. sepsis) or infection-negative origin. It is important to distinguish between septic and non-septic presentations early and reliably, because this has significant therapeutic implications for critically ill patients. We hypothesized that a molecular classifier based on a small number of RNAs expressed in peripheral blood could be discovered that would: 1) determine which patients with systemic inflammation had sepsis; 2) be robust across independent patient cohorts; 3) be insensitive to disease severity; and 4) provide diagnostic utility. The overall goal of this study was to identify and validate such a molecular classifier. Methods and Findings: We conducted an observational, non-interventional study of adult patients recruited from tertiary intensive care units (ICU). Biomarker discovery was conducted with an Australian cohort (n = 105) consisting of sepsis patients and post -surgical patients with infection-negative systemic inflammation. Using this cohort, a four-gene classifier consisting of a combination of CEACAM4, LAMP1, PLA2G7 and PLAC8 RNA biomarkers was identified. This classifier, designated SeptiCyte® Lab, was externally validated using RT-qPCR and receiver operating characteristic (ROC) curve analysis in five cohorts (n = 345) from the Netherlands. Cohort 1 (n=59) consisted of unambiguous septic cases and infection-negative systemic inflammation controls; SeptiCyte® Lab gave an area under curve (AUC) of 0.96 (95% CI: 0.91-1.00). ROC analysis of a more heterogeneous group of patients (Cohorts 2-5; 249 patients after excluding 37 patients with infection likelihood possible) gave an AUC of 0.89 (95% CI: 0.85-0.93). Disease severity, as measured by Sequential Organ Failure Assessment (SOFA) score or the Acute Physiology and Chronic Health Evaluation (APACHE) IV score, was not a significant confounding variable. The diagnostic utility o f SeptiCyte® Lab was evaluated by comparison to various clinical and laboratory parameters that would be available to a clinician within 24 hours of ICU admission. SeptiCyte® Lab was significantly better at differentiating sepsis from infection-negative systemic inflammation than all tested parameters, both singly and in various logistic combinations. SeptiCyte® Lab more than halved the diagnostic error rate compared to PCT in all tested cohorts or cohort combinations. Conclusions: SeptiCyte® Lab is a rapid molecular assay that may be clinically useful in the management of ICU patients with systemic inflammation. SIRS and Sepsis ICU patients, admission samples

Project description:Background: As early and appropriate care of severe septic patients is associated with better outcome, understanding of very first events in the disease process is needed. Pan-genomic analyses offer an interesting opportunity to study global genomic response within the very first hours after sepsis. The objective of this study was to investigate the systemic genomic response in severe Intensive Care Unit (ICU) patients and determine whether patterns of gene expression could be associated with clinical severity evaluated by severity score. Methods: Twenty-eight ICU patients were enrolled at the onset of septic shock. Blood samples were collected within 30 minutes, 24 and 48 hours after shock and genomic response was evaluated using microarrays. The genome wide expression pattern of blood leukocytes was sequentially compared to healthy volunteers and after stratification based on SAPSII score to identify potential mechanisms of dysregulation. Results: Septic shock induces a global reprogramming of the whole leukocyte transcriptome affecting multiple functions and pathways (> 71% of the whole genome was modified). Most altered pathways were not significantly different between SAPSII-high and SAPSII-low groups of patients. However the magnitude and the duration of these alterations were different between these two groups. Importantly, we observed that the more severe patients did not exhibit the strongest modulation. This indicates that some regulation mechanisms leading to recovery seem to take place at early stage. Conclusion: In conclusion, both pro- and anti-inflammatory processes, measured at the transcriptomic level, are induced within the very first hours after septic shock. Interestingly, the more severe patients did not exhibit the strongest modulation. This highlights that, not only the responses mechanisms by themself but mainly their early and appropriate regulation, are crucial for patient recovery. This reinforces the idea that an immediate and tailored aggressive care of patients, aimed at restoring an appropriately regulated immune response, may have a beneficial impact on outcome. Twenty-eight ICU patients were enrolled at the onset of septic shock. Blood samples were collected within 30 minutes, 24 and 48 hours after septic shock and compared to twenty-five healthy volunteers

Project description:BACKGROUND: Despite best supportive intensive care, the development of severe sepsis and septic shock after trauma is still associated with a high mortality rate in intensive care units (ICU). Incomplete knowledge of the pathophysiological and biochemical mechanisms that lead to perturbations of the host system is a major cause for this clinical entity. This lack of knowledge is mirrored in a delay in both diagnosis of sepsis and stratification of patients at risk. The current diagnostic and classification methods do not provide an early and reliable evidence for the development of sepsis and consequently lead to uncertainty in the classification and a delay in the initiation of an adequate therapy. Here, we present a prospective study performed with a well-defined patient cohort suffering from severe multiple trauma (n=85), in which we aimed to uncover the biological reasons why patients with multiple trauma can have dramatically different outcomes after suffering similar traumatic insults. METHODS: We used peripheral whole blood obtained with the PaxGene system at the time of admission to the ICU, i.e., 12 hours after trauma, for transcriptome analysis and monitored clinically the sequence of events from the admission to the ICU, to the very onset of sepsis and finally to the full development of multiple organ dysfunction syndrome (MODS). Study protocols and standard operating procedures (SOPs) regarding ethics, patient recruitment, logistics in sample acquisition and storage, microarray experiments, data analysis and data management have been developed and provided for a high feasibility and reproducibility of the investigations. FINDINGS: The transcriptome analysis from peripheral blood revealed a strong host response upon severe injury and demonstrated the suitability of whole blood in the PaxGene system as a surrogate marker in clinical gene expression studies. It was evident that patients who developed sepsis in the course of the disease (3-5 days after admission) showed an imbalance in the TH1/TH2 response towards a pronounced TH2 response, soon after trauma. The imbalanced TH1/TH2 shift along with inactivation of T cells, neutrophils, monocytes and macrophages might be critical factors for the initiation of a septic program seen in these patients 12 hours after trauma. INTERPRETATION: First, in this study, we provide protocols for logistics and infrastructure that can be applied generally to successfully integrate gene expression analysis studies in a clinical routine and demonstrate that by following these protocols, dedicated and reproducible results can be obtained. Second, we showed that peripheral whole blood is a suitable surrogate marker for the host response as it contains lots of “information” and is easily accessible. Third, we identified mechanisms involved in the perturbations of the immune system that lead to sepsis. Keywords: Prospective study

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Whole blood transcriptome analysis of Septic shock patients according to early therapy response

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