Risk assessment in sepsis: a new prognostication rule by APACHE II score and serum soluble urokinase plasminogen activator receptor

This prospective multicenter study was conducted in 39 departments across Greece from January 2008 to December 2010. The participating departments were 15 ICUs, 18 departments of internal medicine, two departments of pulmonary medicine, three departments of surgery, and one department of urology.

Sepsis patients admitted to the emergency department and sepsis patients presenting during hospitalization in the general ward or in the ICU were eligible. Written informed consent was provided by the patients or by first-degree relatives of patients unable to provide consent. The study protocol was approved by the ethics committees of the participating hospitals. Each patient was enrolled once.

Inclusion criteria were (a) age of at least 18 years; (b) diagnosis of sepsis, severe sepsis, or septic shock; (c) sepsis due to one of the following infections: community-acquired pneumonia (CAP), hospital-acquired pneumonia, ventilator-associated pneumonia, acute pyelonephritis, intra-abdominal infection, or primary bacteremia; and (d) blood sampling within 24 hours from the presentation of signs of sepsis. Six study sites (two ICUs, three departments of internal medicine, and one department of surgery) were selected to be representative of the total enrolment study sites, and agreed to repeat blood sampling on days 3, 7, and 10. First sampling was always done before the administration of any treatment. Exclusion criteria were HIV infection and neutropenia, which was defined as less than 1,000 neutrophils/mm³.

Patients were classified according to standard definitions of sepsis, severe sepsis, and septic shock [9]. Infections were defined according to standard definitions [10‐14]. For each patient, a complete diagnostic work-up was performed. The work-up comprised history, a thorough physical examination, white blood cell count, blood biochemistry, arterial blood gas, blood cultures from peripheral veins and central lines, urine cultures, a chest x-ray, and chest and abdominal computed tomography if appropriate. If necessary, quantitative cultures of tracheobronchial secretions (TBSs) or bronchoalveolar lavage (BAL) were performed and evaluated as previously described [11, 12]. Progress was recorded for 28 days. Clinical and demographic data were recorded in a Case Report Form (CRF). All CRFs were monitored by an independent monitor who was blinded to the study design.

Blood (3 mL) was sampled from every patient. In 367 patients, blood sampling was repeated on days 3, 7, and 10. Blood was collected into sterile and pyrogen- and anticoagulant-free tubes (Vacutainer; Becton Dickinson, Cockeysville, MD, USA). On the same day, tubes were transported by courier to the Laboratory of Immunology of Infectious Diseases of the 4th Department of Internal Medicine at ATTIKON University Hospital (Athens). Tubes were centrifuged, and serum was kept frozen at −70°C until assayed. suPAR was estimated in duplicate by an enzyme-linked immunosorbent assay (suPARnostic™; ViroGates, Lyngby, Denmark) in accordance with the instructions of the manufacturer; the lower detection limit was 1.1 ng/mL. All estimations were performed and reported by two technicians who were blinded to clinical information.

The primary endpoint was to create an accurate prognostication score of death by combining APACHE II score and serum suPAR. The value of this prognostication score was confirmed in a second independent cohort.

The confirmation cohort consisted of pooled results from three prospective studies and involved patients who were at least 18 years old; who had sepsis, severe sepsis, or septic shock; and who were enrolled in three prospective studies of patients with sepsis at the ICU of Karolinska University Hospital in Huddinge, Sweden, during the period of 1998 to 2009. The majority were from the ICU, but five patients were from wards. Demographic characteristics for some of these patients have been published [15‐17]. Sepsis, severe sepsis, and septic shock were defined according to the criteria proposed by the American College of Chest Physicians/Society of Critical Care Medicine [9]. Serum was collected from blood sampled within the first day from diagnosis and was stored at −70°C until analysis. Samples were transported to the same central lab for analysis of suPAR. The technicians did not have access to the clinical information for patients. Based on the results of APACHE II scores and suPAR, a prognostication score was created with which patients in this confirmation cohort were graded for severity. Final matching of prognostication scores and outcome of sepsis was done in Sweden. This ensured that confirmation was conducted in a double-blinded manner.

To create a prognostication rule, the following steps were followed: First, receiver operator curve (ROC) analysis was done with suPAR of day 1 and APACHE II score as independent variables to predict unfavorable outcome. Values of APACHE II score and suPAR with an ROC analysis specificity of above 70% were selected. The latter specificity cutoff was selected since it was considered of importance for risk assessment in sepsis [18]. Second, the importance of the selected cutoffs as independent predictors of unfavorable outcome was defined by step-wise Cox regression analysis. Disease severity, selected cutoffs, and presence of at least one underlying disease were included as independent variables in the equation. Underlying diseases were chronic obstructive pulmonary disorder, diabetes mellitus type 2, chronic renal disease, heart failure, solid tumor malignancy, and chronic intake of corticosteroids since these disease states are widely recognized to affect final outcome. Age, white blood cells, and values of blood gases were not included in the regression analysis, because they were factored into APACHE II. Hazard ratios and 95% CIs were assessed. Third, ROC analysis of the combination of suPAR and APACHE II score was also performed. Given Cook's method of analysis of the role of biomarkers as indexes of disease severity [19], it is highly probable that the ROC generated by the combination of APACHE II score and suPAR does not provide an area under the curve (AUC) superior to that of single APACHE II score or single suPAR. To this end, four strata of severity were generated by using the defined cutoffs of APACHE II score and suPAR. Odds ratios (ORs) and 95% CIs for risk prediction within each stratum were calculated by using Mantel and Haenszel statistics. Comparisons between ORs were done by using Breslow-Day test and Tarone test. Fourth, mortalities between strata were compared by using the chi-squared test and log-rank test. Fifth, comparisons of the risk strata between the study cohort and the confirmation cohort were done by using the chi-squared test. P values of below 0.05 were considered significant.

A total of 1,914 patients were enrolled in the study cohort from a total of 2,145 patients screened for eligibility (Figure 1). All consecutively enrolled patients in the biobank of the Hellenic Sepsis Study Group during the period of January 2008 to December 2010 were included in the present study; 62.2% had sepsis and 37.8% had severe sepsis/septic shock. The most common causes of sepsis were acute pyelonephritis, intra-abdominal infections, and CAP; Gram-negative bacteria were the commonest isolated pathogens. More precisely, positive blood cultures for Escherichia coli were found in 113 patients (5.9%), for Klebsiella pneumoniae in 79 patients (4.1%), for Pseudomonas aeruginosa in 40 patients (2.1%), for Acinetobacter baumannii in 32 patients (1.7%), for other Gram-negative bacteria in 30 patients (1.5%), for Staphylococcus aureus in 21 patients (1.1%), and for Enterococcus spp in 15 patients (0.8%). Positive quantitative urine cultures for E. coli were found in 262 patients (13.7%), for K. pneumoniae in 44 patients (2.3%), for P. aeruginosa in 40 patients (2.1%), for other Gram-negative bacteria in 62 patients (3.3%), and for Enterococcus spp in 29 patients (1.5%). Positive quantitative TBS or BAL cultures for A. baumannii were found in 66 patients (3.6%), for P. aeruginosa in 36 patients (1.9%), for K. pneumoniae in 21 patients (1.1%), and for S. aureus in 16 patients (0.8%).

Among the enrolled patients with sepsis, 1,495 patients survived and 419 died. Patients who died had significantly higher concentrations of suPAR. These patients had a median of 14.06 ng/mL (range of 2.92 to 69.84 ng/mL) in comparison with a median of 9.27 ng/mL (range of 1.14 to 65.00 ng/mL) in survivors (P < 0.0001). To define whether serum suPAR changes over time within survivors and non-survivors, sequential serum measurements were performed for 367 patients, 52 of whom died. At each time point, serum suPAR was significantly higher among non-survivors than among survivors (Figure 2). No significant differences were found after comparing sequential measurements of suPAR separately within survivors and within non-survivors.

ROC analysis indicated that AUCs are much greater for APACHE II score than for suPAR (Figure 3). Coordinate points of ROCs define an APACHE II score of at least 17 as a cutoff and a specificity of greater than 70% to predict death. Similarly, suPAR of at least 12 ng/mL yields a specificity of greater than 70% to predict death.

Given that the AUC of the combination of APACHE II score and suPAR did not differ from that of a single APACHE II score, developing a new prognostication rule required that the above defined cutoffs for APACHE II score and suPAR be independently associated with unfavorable outcome. To investigate this, Cox logistic regression analysis was performed. In the equation, advent of death was set as the dependent variable. Two universally accepted conditions affecting final outcome − namely, the presence of severe sepsis/shock and the presence of at least one underlying disease − were also taken into consideration to try to decipher whether APACHE II score and suPAR may independently prognosticate for unfavorable outcome under the influence of these conditions. Analysis was done in a forward step-wise manner, and results are shown in Table 1. According to this analysis, serum suPAR of at least 12 ng/mL and APACHE II score of at least 17 retained an independent link with unfavorable outcome even when superimposed over the presence of severe sepsis/shock and the presence of underlying diseases. As a consequence, these two cutoffs may be safely used to build a prognostication rule for the assessment of unfavorable outcome in sepsis.

It was then clearly defined that, among patients with an APACHE II score of less than 17 and among patients with an APACHE II score of at least 17, suPAR could significantly indicate those with high risk for death (Table 2). More precisely, OR for death with suPAR of at least 12 ng/mL among patients with an APACHE II score of less than 17 was 3.62; OR was 1.79 with suPAR of at least 12 ng/mL among patients with an APACHE II score of at least 17. The calculated ORs were significantly different (P of comparisons = 0.006 by the Breslow-Day test and P = 0.007 by the Tarone test), indicating that APACHE II score and suPAR were independent prognosticators of unfavorable outcome and should both be used in a prediction model.

With the above cutoff values, four strata of sepsis severity were defined: (i) patients with an APACHE II score of less than 17 and a serum suPAR of less than 12 ng/mL, (ii) patients with an APACHE II score of less than 17 and a serum suPAR of at least 12 ng/mL, (iii) patients with an APACHE II score of at least 17 and a serum suPAR of less than 12 ng/mL, and (iv) patients with an APACHE II score of at least 17 and a serum suPAR of at least 12 ng/mL; 893, 334, 293, and 394 patients ended up in each stratum and had respective mortality rates of 5.5% (n = 49), 17.4% (n = 58), 37.4% (n = 109), and 51.5% (n = 203) (P < 0.0001 within the four defined strata; Figure 4).

This prognostication score corresponds to different grades of sepsis severity, so that more patients with severe sepsis and septic shock had score levels (iii) and (iv) than score levels (i) and (ii). As expected, coexisting illnesses were more common among patients of score levels (iii) and (iv) than score levels (i) and (ii) (Table 3). Sensitivity, specificity, positive predictive value, and negative predictive value of this new score to predict unfavorable outcome change in relation to the strata and are shown in Table 4.

In a further test of the predictive value of this stratification scheme, an independent confirmatory sepsis cohort was used. This cohort included 196 sepsis patients - 108 males and 88 females (P = 0.652 compared with the Greek cohort) - enrolled in Sweden. As shown in Table 5, the Swedish cohort differed considerably from the Greek study cohort in many aspects, including the following:

Respective mortalities of the Swedish patients in relation to the four levels of strata (i), (ii), (iii), and (iv) of the prognostication score were 2.0%, 28.6%, 21.3%, and 33.8% (P < 0.0001 within the four levels). The high mortality in stratum (ii) may be due to small number of patients in this group (n = 21) and chance variations in mortality. Apart from that aberration, the findings validate those from the larger Greek study, even though patients in the two groups are very different (Table 5).