Interleukin 27 as a Sepsis Diagnostic Biomarker in Critically Ill Adults
Wong, Hector R.*†; Lindsell, Christopher J.‡; Lahni, Patrick*; Hart, Kimberly W.‡; Gibot, Sebastien§∥
*Division of Critical Care Medicine, Cincinnati Children’s Hospital Medical Center and Cincinnati Children’s Hospital Research Foundation; and Departments of †Pediatrics; and ‡Emergency Medicine, University of Cincinnati College of Medicine, Cincinnati, OH; and §Réanimation Médicale, Hôpital Central; and ∥INSERM UMR_S 1116, Equipe TREM, Faculté de Médecine, Université de Lorraine, Nancy, France
Received 20 Jun 2013; first review completed 5 Jul 2013; accepted in final form 23 Jul 2013
This study was supported by National Institutes of Health grants RC1HL100474, RO1GM064619, and R01GM099773 and in part by an Institutional Clinical and Translational Science Award, NIH/NCRR 5UL1RR026314.
Address reprint requests to Hector R. Wong, MD, Division of Critical Care Medicine, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45223. E-mail: email@example.com.
HRW conceived and developed the study, obtained funding for the study, directly took part in the analyses, and wrote the manuscript. CJL collaborated in theinitial design of the study and in obtaining funding, oversaw the statistical analyses, and edited the manuscript. PL conducted all biomarker measurements, managed all biological specimens, and edited the manuscript. KWH assisted with statistical analysis and edited the manuscript. SG was the lead investigator for the original study that generated the prospective database used in the current study, assisted with data analysis, and edited the manuscript. All authors read and approved the final manuscript.
Dr. Wong and the Cincinnati Children’s Hospital Research Foundation have submitted a provisional patent application for the use of IL-27 as a sepsis diagnosticbiomarker. The remaining authors have no competing interests to report.
ABSTRACT: Purpose: We previously identified interleukin 27 (IL-27) as a sepsis diagnostic biomarker in critically ill children. The current study tested the performance of IL-27 alone and in combination with procalcitonin (PCT) for diagnosing sepsis in critically ill adults. Methods: Serum samples were made available from a prior prospective study of sepsis biomarkers in critically ill adults. The primary analysis used receiver operating characteristic curves to evaluate the performance of IL-27 and PCT. Secondary analysis explored IL-27 performance in subgroups of patients with sepsis secondary to lung and nonlung sources of infection. The net reclassification improvement was used to estimate the incremental predictive ability of IL-27 compared with PCT alone. Classification and regression tree analysis was used to generate an IL-27- and PCT-based decision tree. Results: There were 145 patients with sepsis and 125 without sepsis. The receiver operating characteristic curve for IL-27 was inferior (area under the curve [AUC], 0.68; 95% confidence interval [CI], 0.62–0.75) to that of PCT (AUC, 0.84; 95% CI, 0.79–0.89). Similar findings were observed when comparing patients with a lung source of infection and those without sepsis. For sepsis patients with a nonlung source of infection, adding IL-27 to PCT improved discrimination (net reclassification improvement = 0.685; P < 0.001). The AUC for the classification and regression tree–derived decision tree was 0.92 (95% CI, 0.88–0.96) and was significantly greater than that of PCT alone. Conclusions: When used in combination with PCT, IL-27 may improve classification of critically ill adults with sepsis secondary to a nonlung source of infection.
There is an unmet need for diagnostic biomarkers of sepsis in critically ill patients (1–3). Procalcitonin (PCT) is currently used as a sepsis diagnostic biomarker, but its performance in critically ill patients has been questioned (4). Consequently, investigators continue to search for additional sepsis biomarkers that can enhance or complement the diagnostic test characteristics of PCT (5–8).
Using genome-wide expression analysis, we identified interleukin 27 (IL-27) as a candidate diagnostic gene for sepsis (9, 10). Interleukin 27 is a heterodimeric cytokine belonging to the IL-6 and IL-12 family of cytokines and is produced by antigen-presenting cells upon exposure to microbial products and inflammatory stimuli (11). Interleukin 27 is a T-cell regulator, having both proinflammatory and anti-inflammatory effects (12, 13), and is rapidly induced in a murine model of septic peritonitis (14). Furthermore, genetic ablation of an IL-27 subunit or neutralization of IL-27 via a soluble IL-27 receptor fusion protein is protective in a murine model of septic peritonitis (14). Thus, it is biologically plausible that IL-27 can serve as a sepsis diagnostic biomarker.
Also plausible in the search for diagnostic biomarkers of sepsis is that different biomarkers are differentially produced depending of the source of infection (15). This would naturally reflect the heterogeneity inherent to the complex syndrome of sepsis. Thus, it is important to consider biomarker performance in different subgroups of patients being evaluated for sepsis secondary to different potential sources of infection.
We have demonstrated that serum IL-27 protein concentrations can differentiate between critically ill children with sterile inflammation and those with laboratory-confirmed bacterial infections (10). In addition, we demonstrated that combining both IL-27 and PCT more accurately identified critically ill children with and without bacterial infection, compared with either biomarker alone. In the current study, we test the ability of IL-27 alone and in combination with PCT to differentiate critically ill adults with and without sepsis. Secondarily, we explored whether the diagnostic accuracy of IL-27 was dependent on the source of infection.
This retrospective diagnostic study used data from a biorepository generated during a prospective study investigating sepsis biomarkers in critically ill adults (5). The enrollment procedures for the study have been previously described in detail (5). Briefly, 300 consecutive patients admitted to the intensive care unit of the University Hospital of Nancy, France, were prospectively enrolled without any exclusion criteria. Adjudication of sepsis or no sepsis classifications was performed by duplicate review of medical records by investigators blinded to biomarker data; consensus was achieved in all cases. All serum samples used in the current study were drawn within 12 h of admission. The original consent included provisions for secondary analyses of biological samples and clinical data, as approved by the institutional review board of the University Hospital of Nancy, France.
Measurement of IL-27 serum protein concentrations
Serum IL-27 protein concentrations were measured for the current study using a magnetic bead multi-plex platform (EMD Millipore Corporation, Billerica, Mass) and a Luminex100/200 System (Luminex Corporation, Austin, Tex), according to the manufacturers’ specifications. Procalcitonin concentrations were measured in the original study using an immunoassay with a sandwich technique and a chemiluminescent detection system (LumiTest; Brahms Diagnostica, Berlin, Germany).
Initially, biomarker data are described using medians and interquartile ranges (IQRs). Biomarker comparisons between groups used the Mann-Whitney U test (SigmaStat Software; Systat Software, Inc, San Jose, Calif). Receiver operating characteristic (ROC) curves and the respective area under the curve (AUC) were constructed and compared using SigmaStat Software. Classification and regression tree analysis was conducted using the Salford Predictive Modeler v6.6 (Salford Systems, San Diego, Calif) (9, 16, 17). Biomarker test characteristics are reported using diagnostic test statistics with 95% confidence intervals computed using the score method as implemented by VassarStats Website for Statistical Computation (18).
The net reclassification improvement (NRI) was used to estimate the incremental predictive ability of IL-27 compared with using PCT alone (19). The NRI ranges between −2 and +2. A score of −2 indicates that all true positives are reclassified as false negatives, and all true negatives are reclassified as false positives, and no false classifications are reclassified as true classifications. Conversely, when the score is 2, adding the information correctly reclassifies every case. The NRI was computed using the R-package Hmisc.
The clinical characteristics of the study subjects were previously published (5). In the original cohort of 300 subjects, there were 154 with sepsis and 146 without sepsis. Remaining serum samples were available for the current study from 145 critically ill adults with sepsis and 125 without sepsis. Among the patients with sepsis, 87 (60%) had a lung source of infection. The next three most common sources of infection were the abdomen (n = 19 [13%]), the urinary tract (n = 11 [8%]), and the central nervous system (n = 8 [6%]). Forty-one sepsis patients (28%) had a documented infection secondary to a gram-negative organism, and 42 sepsis patients (29%) had a documented infection secondary to a gram-positive organism. Fifty-eight sepsis patients (40%) had no organism identified, but met clinical criteria for sepsis. The remaining four sepsis patients had documented infection secondary to either a virus or an intracellular pathogen. Table 1 provides the median (IQR) IL-27 and PCT serum concentrations. Interleukin 27 and PCT serum concentrations were greater in the subjects with sepsis, compared with the subjects without sepsis.
The AUC for the PCT ROC curve (0.840; 95% confidence interval [CI], 0.792–0.888) was significantly greater than that of IL-27 (0.683 [CI, 0.620–0.746], P < 0.001). Tables 2A and 2B provide the diagnostic test characteristics for IL-27 and PCT at different cut points. Procalcitonin performed better than IL-27 as a sepsis diagnostic biomarker at all cut points.
Because the lung was the most common source of infection, we conducted a secondary analysis comparing patients with a lung source of infection (n = 87) and patients with a nonlung source of infection (n = 58). Table 1 provides the median IL-27 and PCT serum concentrations in these two subgroups. Interleukin 27 and PCT concentrations were higher in both sepsis subgroups, compared with the subjects without sepsis.
For differentiating between subjects with a lung source of infection and those without sepsis, the AUC for PCT was significantly greater than that for IL-27 (0.806 [CI, 0.743–0.868] vs. 0.617 [CI, 0.538–0.696], P < 0.001). For differentiating between those with a nonlung source of infection and those without sepsis, the AUC for PCT was also significantly greater than that for IL-27 (0.890 [CI, 0.836–0.944] vs. 0.783 [CI, 0.708–0.859], P = 0.02). However, the AUC for IL-27 in the sepsis subgroup with a nonlung source of infection was improved relative to that for other sepsis patients. Tables 3A and 3B provide the test characteristics for IL-27 and PCT at different cut points in the sepsis subgroup with a nonlung source of infection. Collectively, these secondary analyses suggest that IL-27 expression in sepsis may be dependent on the source of infection and may thus have diagnostic value in sepsis patients with a nonlung source of infection, even if not in patients with a lung source of infection.
Combining IL-27 and PCT
To assess further IL-27 as a sepsis diagnostic biomarker in critically ill patients with a nonlung source of infection, we derived a decision tree incorporating both IL-27 and PCT. Figure 1 shows the derived decision tree, consisting of a very low sepsis probability terminal node (terminal node 1), two high sepsis probability terminal nodes (terminal nodes 5 and 6), and three intermediate sepsis probability nodes (nodes 2–4). Of the 41 cases in the very low sepsis probability node, none (0%) had sepsis. Of the 47 cases in the high sepsis probability nodes, 41 (87%) had sepsis. The proportion with sepsis in the remaining terminal nodes varied from about 9% to about 40%. The diagnostic test characteristics of the decision tree are as follows: sensitivity of 85% (95% CI, 72%–92%), specificity of 86% (95% CI, 78%–91%), positive predictive value (PPV) of 73% (95% CI, 61%–83%), negative predictive value (NPV) of 92% (95% CI, 85–96), positive likelihood ratio (+LR) of 5.9 (95% CI, 3.8–9.1), and negative likelihood ratio (−LR) of 0.2 (95% CI, 0.1–0.3).
Figure 2 shows the ROC curves for the decision tree, PCT alone, and IL-27 alone in the sepsis patients with a nonlung source of infection. The AUC of the decision tree (0.92 [CI, 0.88–0.96]) was significantly greater than that of PCT (P = 0.02) and IL-27 alone (P < 0.001). Furthermore, when adding the IL-27 data to the PCT data, the NRI was 0.69 (0.37–1.00; P < 0.001). This suggests that in critically ill patients with sepsis secondary to a nonlung source of infection, IL-27 may add diagnostic information beyond that provided by PCT alone.
This study represents the first test of IL-27 as a sepsis diagnostic biomarker in critically ill adults. In both the overall sepsis cohort and in the sepsis subgroup with a lung source of infection, the AUC for IL-27 was below 0.7, and the diagnostic test characteristics of IL-27 were inferior to that of PCT. When differentiating between a nonlung source of infection and those without sepsis, however, the AUC for IL-27 approached 0.8. Although the diagnostic test characteristics of IL-27 were also inferior to those of PCT in this subgroup, a decision tree incorporating both IL-27 and PCT suggested an improvement of the overall diagnostic accuracy relative to PCT alone. Compared with PCT alone, when a low IL-27 was measured in conjunction with a low PCT, the negative predictive value for sepsis was correctly increased, and when a high IL-27 was added to a high PCT, the PPV for sepsis was correctly increased. Further support that adding IL-27 to PCT improved discrimination is provided by the NRI. In particular, when differentiating between sepsis patients with a nonlung source of infection and patients without sepsis, a low IL-27 helped to identify more reliably the patients without sepsis when compared with PCT alone. We do note that the NRI has been criticized as having the potential to inflate the incremental prognostic impact of a new biomarker when used in isolation (20). The NRI is this study, however, was consistent with changes in traditional diagnostic test statistics, including the AUC.
The decision tree based on IL-27 and PCT has potential to provide a clinically relevant sepsis probability range, which is otherwise not captured by a single biomarker with a single cut point yielding a dichotomous risk estimate for sepsis. For example, patients in terminal node 1 have extremely low probability for sepsis (0.0%), whereas patients in terminal node 6 have extremely high probability for sepsis (94.1%), thus potentially allowing for biomarker data to directly inform clinical decision making. Alternatively, patients in the remaining terminal nodes have variable, intermediate probabilities for sepsis, thus requiring interpretation and integration of biomarker data with the clinical context for decision making. These assertions require prospective validation.
Our results contrast with our prior study involving critically ill children that demonstrated IL-27 was not only additive, but also outperformed PCT with a specificity and PPV for sepsis of more than 90% (9). Several factors may account for the differences between the pediatric and adult studies. Differences in sample storage conditions could affect the stability of IL-27 and therefore the measurement of IL-27 between the two studies. It is also possible that the IL-27 response of children is different than that of adults, as there are clinical and experimental data demonstrating significantly different responses to inflammatory challenges between developing, pediatric hosts and mature, adult hosts (21–24). We are not aware of any existing data demonstrating a developmental influence on IL-27 expression during infection, and so the potential relationship between developmental age and IL-27 expression is worthy of further investigation. Ultimately, IL-27 may prove to be a more effective sepsis diagnostic biomarker in children than in adults.
It is possible that differences in enrollment criteria for the pediatric and adult cohorts may account for the observed differences in the performance of IL-27 between these two groups. Pediatric patients were required to meet criteria for systemic inflammatory response syndrome (SIRS) and were classified as having sepsis based on laboratory confirmation of a positive culture for known bacterial pathogens, and the majority of these positive cultures were from the blood compartment (9, 25, 26). In contrast, the adult cohort did not require meeting criteria for SIRS (5). Patients in the adult cohort were enrolled consecutively, upon admission to the intensive care unit, irrespective of SIRS criteria, and were subsequently classified as having sepsis based on laboratory and clinical criteria. In addition, a majority of the patients in the adult cohort had a primary lung source of infection. This is an important limitation of our study because it may not be representative of all critically ill populations. For example, it is possible that surgical patients or patients suffering from major trauma may have a lower prevalence of lung infections. Thus, although the pediatric and adult cohorts are both clinically representative, they also reflect different clinical contexts that could influence biomarker performance. This further supports our contention that different biomarkers may have more or less utility in different populations with this highly heterogeneous condition.
In post hoc analyses, we noted that the AUC for IL-27 was 0.768 in subjects with sepsis secondary to a gram-negative organism, whereas the AUC was 0.639 in subjects with sepsis secondary to a gram-positive organism. Thus, future studies of IL-27 as a sepsis diagnostic biomarker should consider the bacterial etiology of sepsis. In addition, future studies may also consider the ability of IL-27 to discriminate between different levels of sepsis severity.
In conclusion, as a general sepsis diagnostic biomarker, IL-27 may not be as effective in critically ill adults as in critically ill children. However, in critically ill adults with sepsis secondary to a nonlung source of infection, IL-27 may add to the sepsis diagnostic accuracy of PCT. Further study of IL-27 as a candidate sepsis biomarker is warranted.
The authors thank the investigators who took part in the original prospective study that generated the database used in the current study.
AUC: area under the curve
IL-27: interleukin 27
IQR: interquartile range
LR: positive likelihood ratio
+LR: positive likelihood ratio
NRI: net reclassification improvement
NPV: negative predictive value
PPV: positive predictive value
ROC: receiver operating characteristic
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Sepsis; diagnosis; biomarkers; decision tree; interleukin 27; procalcitonin
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