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ONLINE DATA SUPPLEMENT to
Prognostic value of secretoneurin in patients with severe sepsis and septic shock: data from the ALBIOS Study
Helge Røsjø MD, PhD1; Serge Masson, PhD2, Pietro Caironi, MD3,4, Mats Stridsberg MD, PhD5; Michela Magnoli, Stat D2; Geir Christensen MD, PhD, MHA6; Gabriella Moise, MD7; Maria Cristina Urbano, MD8; Luciano Gattinoni, MD, FRCP9; Antonio Pesenti, MD10,11; Roberto Latini, MD2; Torbjørn Omland MD, PhD, MPH1; for the ALBIOS Biomarkers Study Investigators
1Division of Medicine, Akershus University Hospital, Lørenskog, Norway and Center for Heart Failure Research, Institute of Clinical Medicine, University of Oslo, Oslo, Norway;
2Department of Cardiovascular Research, IRCCS – Istituto di Ricerche Farmacologiche
“Mario Negri”, Milan, Italy; 3Anestesia e Rianimazione, Azienda-Ospedaliero Universitaria S. Luigi Gonzaga, Orbassano, Italy; 4Dipartimento di Oncologia, Università degli Studi di Torino, Turin, Italy; 5Department of Medical Sciences, Uppsala University, Uppsala, Sweden;
6Institute for Experimental Medical Research, Ullevål and Center for Heart Failure Research, Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 7Ospedale Città di Sesto San Giovanni, Sesto San Giovanni, Italy; 8Ospedale della Misericordia, Grosseto, Italy;
9Department of Anesthesiology, Emergency and Intensive Care Medicine, University of Göttingen, Germany.10Dipartimento di Anestesia, Rianimazione ed Emergenza Urgenza, Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy;
11Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, Italy
2 SUPPLEMENTARY MATERIAL
ALBIOS Study
The total study cohort of ALBIOS included 1818 patients age 18 y or older who met clinical criteria for severe sepsis or septic shock within 24 h of hospital admittance or anytime during the Intensive Care Unit (ICU) stay (1). Eight patients were excluded from the analysis (2 patients in the albumin group owing to withdrawal of consent, and 5 in the albumin group and 1 in the crystalloid group owing to a randomization error). Severe sepsis was defined as proven or suspected infection, two or more signs of the systemic inflammatory response syndrome, and at least one acute sepsis-related organ dysfunction, as defined by the Sequential Organ Failure Assessment (SOFA) score (1). Shock on study inclusion was defined as a score of 3 or 4 on the SOFA cardiovascular component. SOFA scores were calculated on a daily basis and we also calculated the Simplified Acute Physiology Score (SAPS) II score (2)after 24 h in the ICU.
In the first 24 h after study inclusion, fluids were administered to both groups according to the protocol for early-goal directed therapy (3). In patients randomized to 20% albumin, albumin administration was targeted at a serum albumin level ≥30 g/L for. Demographic, clinical and laboratory data were collected on a daily basis and microbiological sampling was performed according to local protocols. All treatments, except fluid administration, were left to the discretion of the attending physicians in the different ICUs that participated in the ALBIOS trial, according to the international guidelines for the treatment of sepsis.
Biomarker analyses
Blood samples were centrifuged and allocated, and frozen plasma samples were shipped to a central biorepository and stored at -70° C. We used the Elecsys proBNP assay (Roche
Diagnostics, Penzberg, Germany) for N-terminal pro-B-type natriuretic peptide (NT-proBNP) measurements and the Elecsys TNT hs STAT assay (Roche Diagnostics) for high-sensitivity cardiac troponin T (hs-cTnT) measurements (4).
Statistical analysis
Differences in clinical characteristics and hemodynamic goals across tertiles of SN concentration were compared by chi-square test of Fisher’s exact test for categorical variables; for continuous variables, we used analysis of variance (ANOVA) or the non- parametric Kruskal-Wallis test. We calculated Spearman rank correlation coefficients
between continuous variables and SN levels on day 1. Moreover, to identify variables related to high SN levels on day 1, considered on a natural logarithm scale, a stepwise multivariable linear regression model was performed including all baseline characteristics significantly associated with secretoneurin at a p level <0.05. Non-parametric analysis of variance on ranks of SN concentrations was used to assess (1) the influence of prevalent shock over time in the overall population and (2) the effect of the randomized treatments (albumin vs. crystalloids)
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over time separately in patients with or without septic shock. Only patients with SN measurements at all three time points were considered for this analysis.
The relationship between SN concentrations at each time point (days 1, 2 and 7) with ICU mortality and 90-day mortality was first described by median SN levels (Q1-Q3) and
compared with Kruskal-Wallis test. Then, a univariate logistic regression model was done to evaluate the prognostic value of SN, considered as a continuous variable after natural
logarithm transformation. Finally, multivariable logistic regression models were performed to evaluate the independent prognostic value of SN, adjusting for the variables associated with the outcome of interest. In addition to clinical variables, we included hs-cTnT and NT-
proBNP levels into multivariable models as both of these established cardiac biomarkers also were associated with clinical outcomes. We assessed the prognostic utility of SN separately in the subgroups of patients with severe sepsis with or without septic shock at study entry.
Measures of discrimination ability were calculated, in patients with septic shock, as (1) the area under the ROC curve (AUROC) independently for each circulating marker (SN, NT- proBNP and hs-cTnT), and (2) the continuous Net Reclassification Improvement (cNRI), obtained after adding SN concentrations to the multivariate models described above. Kaplan- Meier survival analysis was performed by tertiles of SN concentrations on day 1 in the subgroups of patients with severe sepsis with or without shock. Normally distributed changes in SN concentrations on day 2 from day 1, and on day 7 from day 2, were expressed as the difference in log-transformed levels and analysed with univariate and multivariate logistic regression model to assess the relationship with ICU mortality and 90-day mortality.
A two-sided p value of <0.05 was deemed statistically significant. All statistical analyses were performed with SASsoftware (SAS Institute, Cary, NC, USA) version 9.4.
4 REFERENCES
1. Vincent JL, Moreno R, Takala J, et al. The SOFA (Sepsis-related Organ Failure
Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Medicine 1996;22:707-10.
2. Le Gall JR, Lemeshow S, Saulnier F. A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study. JAMA 1993;270:2957-63.
3. Dellinger RP, Levy MM, Rhodes A, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Medicine 2013;39:165-228.
4. Masson S, Caironi P, Fanizza C, et al. Sequential N-terminal pro-B-type natriuretic peptide and high-sensitivity cardiac troponin measurements during albumin replacement in patients with severe sepsis or septic shock. Critical Care Medicine 2016;44:707-16.
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Supplementary Table 1. Comparison between patients with SN measured on day 1 (n=958) and the total ALBIOS study cohort (n=1810).
ALBIOS population (n=1810)
Present study (SN population) (n =958)
Characteristics Albumin
(n=903)
Crystalloids (n=907)
Albumin (n=474)
Crystalloids (n=484)
Age – year 70 [57-77] 69 [59-77] 70 [57–78] 70 [59–78]
Female sex – No. (%) 360 (39.9) 357 (39.4) 198 (41.8) 197 (40.7)
Reason for admission to ICU- No. (%)
Medical 511 (56.6) 518 (57.1) 273 (57.6) 267 (55.2)
Elective surgery 69 (7.6) 58 (6.4) 37 (7.8) 30 (6.2)
Emergency surgery 323 (35.8) 331 (36.5) 164 (34.6) 187 (38.6)
Preexisting conditions – No. (%)
Liver disease 13 (1.4) 14 (1.5) 7 (1.5) 7 (1.5)
COPD 113 (12.5) 108 (11.9) 63 (13.3) 57 (11.8)
Chronic renal failure 44 (4.9) 32 (3.5) 25 (5.3) 17 (3.5)
Immunodeficiency 115 (12.7) 128 (14.1) 60 (12.7) 64 (13.2)
Congestive or ischemic heart disease 149 (16.5) 165 (18.2) 77 (16.2) 87 (18.0)
SAPS II score 48 [37–59] 48 [37–60] 46 [36-55] 46 [35-57]
Physiological /Laboratory variables
Heart rate – beats/min 105±22 106±20 103±20 105± 21
Mean arterial pressure – mmHg 74±16 73±15 75±16 74±14
Central venous pressure – mmHg 10.0±4.9 9.8±4.7 10.1±4.7 9.8±4.6
Urine output – ml/hr 50 [20–100] 50 [25–100] 60 [27–100] 60 [30–100]
Lactate – mmol/liter 2.3 [1.4–4.2] 2.5 [1.6–4.3] 2.3 [1.4–4.0] 2.3 [1.5–4.0]
Central venous oxygen saturation 73 [65–79] 73 [68–80] 74 [66–79] 74 [69–80]
SOFA score [at baseline] 8 [6–10] 8 [5–10] 7 [5-10] 7 [5-9]
Shock – No. (%) 565 (62.6) 570 (62.8) 273 (57.6) 267 (55.2)
Mechanical ventilation – No. (%) 709 (78.5) 737 (81.3) 371 (78.3) 389 (80.4)
Death at 28 days – no./total No. (%) 285/895 (31.8) 288/900 (32.0) 135/468 (28.9) 128/481 (26.6) Death at 90 days – no./total No. (%) 365/888 (41.1) 389/893 (43.6) 185/467 (39.6) 184/478 (38.5)
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Supplementary Table 2. Characteristics of the ALBIOS biomarker substudy with patients
stratified according to SN tertiles measured on day 1 after randomization
Characteristics SN on day 1 (pmol/L)
All T1
(11.1-174.0)
T2 (175.0-236.0)
T3
(237.0-2791.0) P
No. (%) 958 (100) 319 (33.3) 319 (33.3) 320 (33.4)
Age – year 70 [58–78] 66 [49–76] 71 [61–78] 71 [62–80] < 0.0001
Female sex – no. (%) 395 (41.2) 132 (41.4) 143 (44.8) 120 (37.5) 0.17
Body Mass Index – kg/m2 26.43±5.57 26.33±6.03 26.73±5.59 26.23±5.04 0.48
Reason for admission to ICU - no. (%) 0.16
Medical 540 (56.4) 175 (54.9) 167 (52.4) 198 (61.9)
Elective surgery 67 (7.0) 23 (7.2) 26 (8.2) 18 (5.6)
Emergency surgery 351 (36.6) 121 (37.9) 126 (39.5) 104 (32.5)
Preexisting conditions – no. (%)
Liver disease 14 (1.5) 6 (1.9) 3 (0.9) 5 (1.6) 0.60
COPD 120 (12.5) 35 (11.0) 42 (13.2) 43 (13.4) 0.59
Chronic renal failure 42 (4.4) 4 (1.3) 9 (2.8) 29 (9.1) <.0001
Immunodeficiency 124 (12.9) 43 (13.5) 37 (11.6) 44 (13.8) 0.68
Congestive or ischemic heart disease 164 (17.1) 39 (12.2) 56 (17.6) 69 (21.6) 0.01
SAPS II score 46.7±15.6 40.5±13.4 45.9±14.5 53.9±9
<0.0001 Physiological /Laboratory variables
Heart rate – beats/min 104±21 102±19 104±22 105±21 0.11
Mean arterial pressure – mmHg 75±15 76±14 76±16 72±15 0.0004
Mean arterial pressure after 6 hrs – mmHg 78±13 80±13 79±14 76±14 0.0007
Central venous pressure – mmHg 9.95±4.64 9.50±4.15 9.90±4.61 10.46±5.09 0.04
PaO2 /FiO2 190 [130–270] 190 [130–270] 190 [130–270] 200 [130–280] 0.51
Urine output – ml/hr 60 [30–100] 70 [50 –100] 60 [32 –100] 50 [10–90] <0.0001
Lactate – mmol/liter 2.3 [1.4–4.0] 2.0 [1.3–3.5] 2.5 [2.2–1.4] 2.7 [1.7–5.1] <0.0001
Serum albumin – g/liter 24.44±6.25 24.54±6.35 24.54±5.98 24.21±6.42 0.78
Hemoglobin – g/dl 10.92±1.97 11.18±1.95 10.91±2.00 10.66±1.92 0.004
Serum creatinine – mg/dL 1.5 [0.9–2.5] 1.0 [0.7–1.5] 1.4 [0.9–2.2] 2.6 [1.6–3.8] <0.0001 White blood cells – 103/mm3 12.0 [5.7–18.6] 11.7 [5.5–18.7] 12.1[6.6–18.8] 12.1 [5.6–17.8] 0.60
Central venous oxygen saturation 72.60±9.80 74.03±8.99 72.08±9.71 71.71±10.49 0.01
Serum bilirubin (mg/dL) 0.8 [0.5–1.5] 0.8 [0.5–1.3] 0.8 [0.5–1.6] 0.9 [0.6–1.8] 0.03 Platelet count (109/L) 166 [102–237] 173 [113–240] 170 [106–241] 145 [83–233] 0.007 NT-proBNP – median [Q1-Q3] 4399 [1313-13837] 2063 [738-5724] 3928 [1449-10371] 10734 [3791-26838] < 0.0001
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hs-cTnT – median [Q1-Q3] 50.4 [21.7-133.5] 31.5 [14.0-86.0] 44.8 [21.0-112.7] 88.4 [41.4-217.8] < 0.0001
SOFA score [at baseline] 7 [5-10] 6 [4-8] 7 [5-9] 9 [7-11] <0.0001
Shock – no. (%) 540 (56.4) 161 (50.5) 179 (56.1) 200 (62.5) 0.01
Mechanical ventilation – no. (%) 760 (79.3) 249 (78.1) 258 (80.9) 253 (79.1) 0.67
Randomized to Albumin arm – no. (%) 474 (49.5) 157 (49.2) 154 (48.3) 163 (50.9) 0.79
Antibiotics at the time of randomization –
no (%) 898 (93.7) 298 (93.4) 297 (93.1) 303 (94.7) 0.68
Antibiotics at 6 hours after randomization –
no (%) 954 (99.6) 318 (99.7) 317 (99.4) 319 (99.7) 0.85
Appropriateness of antibiotic therapy at Day 1 after randomization according to site culture – no (%)
422/558 (75.6) 131/171 (76.6) 138/186 (74.2) 153/201 (76.1) 0.85 Appropriateness of antibiotic therapy at
Day 1 after randomization according to blood culture – no (%)
211/259 (81.5) 53/69 (76.8) 72/85 (84.7) 86/105 (81.9) 0.45
ANOVA or Kruskal-Wallis test for continuous variables, chi-square or Fisher tests for categorical variables.
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Supplementary Table 3. Correlation coefficients between SN levels measured on day 1 after randomization and continuous laboratory or clinical variables
Variables rho P
Serum creatinine – mg/dL 0.51 <0.0001
NT-proBNP – ng/L 0.40 <0.0001
SAPS II score 0.37 <0.0001
SOFA score [at baseline] 0.35 <0.0001
hs-cTnT – ng/L 0.31 <0.0001
Urine output – ml/hr -0.22 <0.0001
Lactate – mmol/liter 0.17 <0.0001
Age – year 0.16 <0.0001
Mean arterial pressure after 6 hrs – mmHg -0.16 <0.0001
Mean arterial pressure – mmHg -0.14 <0.0001
Serum bilirubin (mg/dL) 0.11 0.001
Platelet count (109/L) -0.11 0.001
Hemoglobin – g/dL -0.10 0.002
Central venous pressure – mmHg 0.09 0.009
Central venous oxygen saturation (%) -0.09 0.01
Heart rate – beats/min 0.07 0.02
Serum albumin – g/liter -0.03 0.33
PaO2 /FiO2 0.02 0.49
Body-mass index – kg/m2 0.008 0.82
White blood cells – 103/mm3 -0.002 0.94
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Supplementary Table 4. Variables associated with increasing SN levels on day 1
Linear regression model in which the dependent variables is the natural logarithm transformed SN levels and the following covariates: age, chronic renal failure, congestive or ischemic heart disease, mean arterial pressure, central venous pressure, urine output, serum lactate, hemoglobin, serum creatinine, central venous oxygen saturation, serum bilirubin, platelets count, shock, NT-proBNP and hs-cTnT on day 1.
Variable Parameter
Estimate
Standard Error
t value P
Serum creatinine (mg/dl) 0.080 0.009 9.28 <0.0001 NT-proBNP (ng/L) 0.000006 0.000001 4.42 <0.0001 Serum bilirubin (mg/dL) 0.024 0.009 2.70 0.007 Serum lactate (mmol/L) 0.014 0.005 2.68 0.008
Age (year) 0.0018 0.0009 2.05 0.04
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Supplementary Table 5. Achievement of hemodynamic goals during the first 6-24 h and day 1 concentrations of SN
Characteristics SN on day 1
All T1 (11-174) T2 (175-236) T3 (237-2791) P
Central venous pressure after 6 hours – no. (%) 0.17
< 8 mmHg 205/907 (22.6) 82/308 (26.6) 62/301 (20.6) 61/298 (20.5)
8-12 mmHg 398/907 (43.9) 130/308 (42.2) 142/301 (47.2) 126/298 (42.3)
> 12 mmHg 304/907 (33.5) 96/308 (31.2) 97/301 (32.2) 111/298 (37.3)
Mean arterial pressure after 6 hours ≥ 65 mmHg – no. (%) 822/958 (85.8) 286/319 (89.7) 277/319 (86.8) 259/320 (80.9) 0.006 Central venous oxygen saturation after 6 hours ≥ 70% - no. (%) 612/811 (75.5) 229/275 (83.3) 188/268 (70.2) 195/268 (72.8) 0.0008 Lactate at day 1 < 2 mmol/liter – no. (%) 562/923 (60.9) 218/307 (71.0) 194/306 (63.4) 150/310 (48.4) <.0001 Vasoactive drugs at day 1 – no. (%)
Dopamine 234/948 (24.7) 71/318 (22.3) 77/316 (24.4) 86/314 (27.4) 0.33
Norepinephrine 514/948 (54.2) 149/318 (46.9) 163/316 (51.6) 202/314 (64.3) <.0001
Epinephrine 57/948 (6.0) 10/318 (3.1) 15/316 (4.8) 32/314 (10.2) 0.0005
Dobutamine 134/948 (14.1) 29/318 (9.1) 43/316 (13.6) 62/314 (19.8) 0.0006
Vasopressin 9/948 (0.9) 4/318 (1.3) 1/316 (0.3) 4/314 (1.3) 0.36
Two or more vasoactive drugs at day 1 – no. (%) 265/948 (28.0) 58/318 (18.2) 81/316 (25.6) 126/314 (40.1) <.0001 Doses of vasoactive drugs at day 1 - µg/kg/min
Dopamine 7.44±3.94 6.72±3.29 7.91±4.46 7.61±3.92 0.17
Norepinephrine 0.28±0.30 0.24±0.26 0.28±0.28 0.30±0.35 0.21
Epinephrine 0.13±0.12 0.11±0.07 0.09±0.06 0.16±0.15 0.16
Inotropic score on day 1 8 [0–21] 5 [0–18] 8 [0–21] 10 [0–29] <.0001
Mean arterial pressure at day 1 ≥ 65 mmHg – no. (%) 852/948 (89.9) 298/318 (93.7) 294/316 (93.0) 260/314 (82.8) <.0001
The inotropic score was calculated as dopamine dose on day 1 + (epinephrine dose at day 1)*100 + (norepinephrine dose at day 1)*100 (7).
ANOVA for continuous variables, Chi-square test for categorical variables.
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Supplementary Table 6. Logistic regression models to assess interaction between SN concentrations on day 1 and septic shock for ICU and 90- day mortality
ICU mortality: analysis of Maximum Likelihood Estimates
Parameter DF Estimate Standard
Error
Wald Chi-Square
Pr > ChiSq
SN on day 1 (ln transformed) 1 1.1985 0.2040 34.5160 <.0001
Prevalent shock 1 -3.1568 1.1023 8.2011 0.004
Interaction between shock and SN on day 1 1 0.6138 0.2040 9.0522 0.003
90-day mortality: analysis of Maximum Likelihood Estimates
Parameter DF Estimate Standard
Error
Wald
Chi-Square Pr > ChiSq SN on day 1 (ln transformed) 1 1.2290 0.1964 39.1707 <.0001
Prevalent shock 1 -2.0185 1.0526 3.6775 0.06
Interaction between shock and SN on day 1 1 0.4132 0.1964 4.4281 0.04
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Supplementary Table 7. Associations between changes in SN levels during ICU stay and clinical outcomes
Outcome Patients Changes from day 1 to day 2 Changes from day 2 to day 7
Univariate Multivariable Univariate Multivariable
No. events / no.
patients (%) OR [95%CI] p OR [95%CI] p No. events / no.
patients (%) OR [95%CI] p OR [95%CI] p
ICU mortality Severe sepsis 84/384 (21.9) 0.99 [0.49-2.00] 0.97 1.18 [0.51–2.71] 0.69 61/320 (19.1) 0.50 [0.21-1.21] 0.12 0.51 [0.19–1.41] 0.20 Shock 132/486 (27.2) 1.40 [0.71-2.74] 0.33 2.09 [0.93–4.67] 0.07 87/395 (22.0) 0.61 [0.27-1.39] 0.24 0.61 [0.24–1.53] 0.29 90 day mortality Severe sepsis 123/375 (32.8) 0.71 [0.37-1.36] 0.30 0.90 [0.40–1.99] 0.78 98/311 (31.5) 0.66 [0.31-1.40] 0.28 1.00 [0.40–2.49] 0.99 Shock 202/483 (41.8) 1.56 [0.84-2.88] 0.16 1.73 [0.84–3.53] 0.14 151/390 (38.7) 0.51 [0.25-1.05] 0.07 0.50 [0.21–1.19] 0.12
Changes from day 1 to day 2 or 7 were considered as difference in natural logarithm concentration from day x to day y [ln(SNday y) – ln(SNday x)].
Covariates included in the multivariable analyses were the same as in the multivariate models #1 reported below Table 2.
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Supplementary Figure 1. Secretoneurin concentration on day 1 by quintiles of overall SOFA score and by organ-specific SOFA sub-scores on day 1
For the overall SOFA score: Q1= 0–4, Q2= 5–6, Q3= 7–8, Q4= 9–10, Q5= 11–18.
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Supplementary Figure 2. Time-course of SN concentration by shock and randomized treatment
SN concentrations on days 1, 2 and 7 are reported in patients with the three time-points available, 311 patients with severe sepsis without shock (151 randomized to albumin, 160 to crystalloids) and 377 with septic shock (190 randomized to albumin, 187 to crystalloids). Data shown as median [Q1-Q3] after stratification for sepsis severity (A) or sepsis severity and randomized treatment (B). Non-parametric analysis of variance were done on ranks of SN concentrations. In all patients: time p=1.00, shock p=0.0004, interaction time by shock p=0.62; in patients with severe sepsis without shock: time p=1.00, treatment p=0.47, interaction time by treatment p=0.79; in patients with septic shock: time p=1.00, treatment p=0.24, interaction time by treatment p=0.91. * P<0.05 vs. severe sepsis at corresponding time point by Kruskal-Wallis test.
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Supplementary Figure 3. Reclassification of mortality by SN levels on day 2 in patients with septic shock
SN levels on day 2 reclassified a significant proportion of patients with septic shock into their correct risk strata when added on top of the basic risk model (Model #1 in Table 2), both for ICU mortality (top panels: NRI 0.30 [95% CI 0.10-0.51], p=0.004; NRI events= 12%, p=0.21;
NRI non-events= 18%, p=0.0006) and 90 day mortality (lower panels: NRI 0.22 [0.05-0.42], p=0.01; NRI events = 10%, p=0.13; NRI non-events= 12%, p=0.05).
