Supplementary Appendix
Circulating presepsin (soluble CD14 subtype) as a marker of host response in patients with severe sepsis or
septic shock. Data from the multicenter, randomized ALBIOS trial.
Serge Masson; Pietro Caironi; Caterina Fanizza; Ralf Thomae; Roberto Bernasconi, Andrea Noto, Roberto Oggioni,
Giovanni Stefano Pasetti, Marilena Romero; Gianni Tognoni; Roberto Latini; Luciano Gattinoni; on behalf of the
ALBIOS Study Investigators§
1
Supplementary Methods
Study design, data collection, and definitions
Inclusion and exclusion criteria, and study design of the ALBIOS trial have been published elsewhere [1], and
are briefly summarized here. Patients aged 18 years or older who met clinical criteria for severe sepsis within the
previous 24 hours and at any time during ICU stay, were enrolled in the ALBIOS trial. During the early phase of
volume resuscitation (6-24 hours after study enrollment), fluids were administered in both groups according to the
“early goal-directed therapy” protocol [2]. Primary outcome was all-cause mortality 28 days after randomization, and
principal secondary outcome was all-cause mortality at 90 days. Additional secondary outcomes included the number
and severity of organ dysfunctions, respectively defined as the number of both novel organ dysfunction and organ
failure occurring during study period (based upon the SOFA score), and the average of the daily SOFA scores for each
individual patient during the study period, ICU and hospital length of stay. Tertiary outcomes included the use of renal
replacement therapy, the incidence of acute kidney injury, the duration of mechanical ventilation, the time to suspension
of vasopressor or inotropic agents, the clinical resolution and duration of the primary infection, and the incidence of
secondary infections, all during the study period. Clinical resolution of the primary infection, if any, was established by
the treating physician according to standard clinical signs. Acquired secondary infections were defined as new
infections other than the primary infection responsible for the development of severe sepsis, arising during the study,
and evaluated by the treating physician. Immunodeficiency at the time of randomization was defined as the exposition
to immunosuppressive therapy before study enrollment (such as immunosuppressive agents, steroids, chemotherapy,
and radiative therapy), or the presence of a co-morbidity able to suppress defenses of the immune system (neoplasia,
blood tumor, AIDS, etc.).
Demographic, clinical, laboratory and microbiological data were collected on a daily basis. Overall severity of
the systemic illness was assessed at study entry with the Simplified Acute Physiology Score SAPS II [3]. Organ
function was rated daily using the Sequential Organ Failure Assessment (SOFA) score [4], including sub-scores from 0
to 4 for five components (lungs, coagulation, liver, circulation, kidney), with high scores indicating a high degree of
organ dysfunction. Microbiological sampling, as well as routine surveillance cultures, were done at the discretion of the
attending physician, and according to standard practice in each center. All the treatments applied with the exception of
fluid management were at the discretion of the attending physician [1], who usually did not coincide with the local
investigator.
2
Inclusion in the pre-defined biomarkers study
A subset of 997 patients, recruited in 40 centers, participated to the predefined biomarkers sub-study. About
70% of the patients enrolled in the original ALBIOS trial from each of those centers participated also into the biomarker
sub-study. Venous blood samples were serially collected 1, 2 and 7 days after enrolment (or at ICU discharge,
whichever came first), centrifuged, and plasma was shipped on dry ice to a central repository and stored at -70°C until
assayed.
Additional statistical methods
Multivariable linear regression analysis with backward elimination was performed to identify the clinical
determinants independently associated with a high log-transformed concentration of presepsin on day 1. The analysis
included continuous variables (age, heart rate, mean arterial pressure, central venous pressure, urine output, lactate,
serum albumin, hemoglobin, serum creatinine, white blood cells, serum bilirubin, platelet count) and categorical
variables (chronic renal failure, immunodeficiency, shock, primary site of infection, type of infection according to site
culture, type of infection according to blood culture).
The relation between presepsin concentration and mortality was first assessed with univariate logistic models
entering log-transformed concentration of the biomarker as a continuous variable. The following covariates were then
introduced into a multivariable model if they had a simple association with outcome with p<0.20: age, sex, body mass
index, reason for admission in ICU, SAPS II score, SOFA score at baseline, appropriate antibiotic therapy, heart rate,
mean arterial pressure at baseline or after 6 hours, central venous pressure, PaO 2/FiO2, urine output, serum
concentration of lactate, albumin, hemoglobin, creatinine or bilirubin, platelet count, mechanical ventilation, COPD,
chronic renal failure, immunodeficiency, congestive or ischemic heart disease, liver disease, shock.
The relations between tertiary outcomes and presepsin concentration were analyzed with a multivariate logistic
regression model and a linear regression model for categorical and continuous variables, respectively. The variables
included in the model were age, immunodeficiency, shock, mechanical ventilation and SOFA score at baseline.
Statistical analysis was done using SAS software, version 9.3 (SAS Institute). A two-sided p value of <0.05 was
deemed statistically significant.
3
Supplementary References
1. Caironi P, Tognoni G, Masson S, Fumagalli R, Pesenti A, et al (2014). Albumin Replacement in Patients with Severe
Sepsis or Septic Shock. N Engl J Med 370: 1412-1421.
2. Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, et al (2001). Early goal-directed therapy in the treatment of
severe sepsis and septic shock. N Engl J Med 345: 1368-1377.
3. Le Gall JR, Lemeshow S, Saulnier F (1993). A new Simplified Acute Physiology Score (SAPS II) based on a
European/North American multicenter study. JAMA 270: 2957-2963.
4. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonça A, et al (1996). 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 Med 22: 707-710.
4
Supplementary Table 1: Presepsin levels on day 1 and infection
Variable
Primary site of infection
Lung
Abdomen
Urinary tract
Other site
More than one site
Type of infection according to site culture
Bacterial infection
Purely gram-negative bacteria
Purely gram-positive bacteria
Mixed bacteria
Culture negative
Culture not obtained
Other infection
Type of infection according to blood culture
Bacterial infection
Purely gram-negative bacteria
Purely gram-positive bacteria
Mixed bacteria
Culture negative
Culture not obtained
Other infection
No. (%)
332 (34.7)
94 (9.8)
91 (9.5)
334 (34.9)
107 (11.2)
411 (42.9)
211 (22.0)
133 (13.9)
67 (7.0)
314 (32.8)
60 (6.3)
173 (18.0)
229 (23.9)
98 (10.2)
112 (11.7)
19 (2.0)
581 (60.6)
106 (11.1)
42 (4.4)
Presepsin
P
(median [Q1-Q3], ng/L)
<0.0001
958 [518-1832]
1200 [630-3309]
1224 [553-2821]
762 [397-1340]
982 [589-2405]
0.0003
1025 [558-2197]
1062 [557-2319]
972 [577-1863]
1097 [558-2160]
773 [388-1767]
735 [443-1529]
943 [549-1962]
<0.0001
1178 [657-2623]
1398 [763-2774]
963 [559-2297]
1887 [813-3714]
868 [454-1589]
850 [357-1846]
1141 [549-2440]
5
Supplementary Table 2: Presepsin levels on day 1 and achievement of hemodynamic goals
Presepsin on Day 1
All
Tertile 1
(<597 ng/L)
Tertile 2
(597-1397 ng/L)
Tertile 3
(>1397 ng/L)
No. (%)
At randomization
Mean arterial pressure ≥ 65 mmHg – no. (%)
Vasoactive drugs – no. (%)
Dopamine
Norepinephrine
Epinephrine
Dobutamine
Vasopressin
958 (100.0)
320 (33.4)
320 (33.4)
318 (33.2)
719 (75.1)
609 (63.6)
248 (25.9)
462 (48.2)
44 (4.6)
63 (6.6)
10 (1.0)
266 (83.1)
168 (52.5)
70 (21.9)
123 (38.4)
9 (2.8)
8 (2.5)
2 (0.63)
242 (75.6)
219 (68.4)
85 (26.6)
170 (53.1)
15 (4.7)
29 (9.1)
5 (1.6)
211 (66.4)
222 (69.8)
93 (29.3)
169 (53.1)
20 (6.3)
26 (8.2)
3 (0.9)
<0.0001
<0.0001
0.03
0.0002
0.04
0.004
0.7
Two or more vasoactive drugs – no (%)
Doses of vasoactive drugs - µg/kg/min
201 (21.0)
41 (12.8)
79 (24.7)
81 (25.5)
<0.0001
8.27 ± 4.52
0.28 ± 0.27
0.15 ± 0.21
8.25 ± 3.96
0.25 ± 0.26
0.15 ± 0.18
7.52 ± 4.33
0.28 ± 0.27
0.11 ± 0.08
8.96 ± 5.00
0.29 ± 0.27
0.18 ±0.28
0.70
0.05
0.69
822 (85.8)
298 (93.1)
281 (87.8)
243 (76.4)
<0.0001
205 (22.6)
398 (43.9)
304 (33.5)
612 (75.5)
79 (25.7)
140 (45.6)
88 (28.7)
217 (77.8)
71 (23.6)
137 (45.5)
93(30.9)
213 (77.7)
55 (18.4)
121 (40.5)
123 (41.1)
182 (70.5)
852 (89.9)
562 (60.9)
652 (68.8)
234 (24.7)
514 (54.2)
57 (6.0)
134 (14.1)
9 (0.9)
304 (95.6)
237 (76.7)
179 (56.3)
64 (20.1)
131 (41.2)
10 (3.1)
28 (8.8)
1 (0.3)
289 (91.2)
185 (59.9)
228 (71.9)
74 (23.3)
178 (56.2)
19 (6.0)
49 (15.5)
4 (1.3)
259 (82.8)
140 (45.9)
245 (78.3)
96 (30.7)
205 (65.5)
28 (9.0)
57 (18.2)
4 (1.3)
<0.0001
<0.0001
<0.0001
0.002
<0.0001
0.002
0.0007
0.21
265 (28.0)
51 (16.0)
88 (27.8)
126 (40.3)
<0.0001
7.44 ± 3.95
0.28 ± 0.30
0.13 ± 0.12
6.82 ± 3.38
0.24 ± 0.27
0.08 ± 0.05
7.23 ± 3.63
0.29 ± 0.33
0.11 ± 0.07
8.01 ± 4.46
0.28 ± 0.31
0.16 ± 0.16
0.15
0.14
0.13
Dopamine
Norepinephrine
Epinephrine
After 6 hours
Mean arterial pressure ≥ 65 mmHg – no. (%)
Central venous pressure – no. (%)
< 8 mmHg
8-12 mmHg
> 12 mmHg
Central venous oxygen saturation ≥ 70% - no.
(%)
On day 1
Mean arterial pressure ≥ 65 mmHg – no. (%)
Lactate < 2 mmol/L – no (%)
Vasoactive drugs – no. (%)
Dopamine
Norepinephrine
Epinephrine
Dobutamine
Vasopressin
Two or more vasoactive drugs – no (%)
Doses of vasoactive drugs - µg/kg/min
Dopamine
Norepinephrine
Epinephrine
P across
tertiles
0.001
0.05
P for trend across tertiles were evaluated with the Cochran-Armitage test or one-way analysis of variance with tertiles
of presepsin as a continuous variable.
6
Supplementary Table 3: Presepsin and clinical outcomes
Outcome
Presepsin at day 1
All
Tertile 1
(<597 ng/L)
Tertile 2
(597-1397 ng/L)
Tertile 3
(> 1397 ng/L)
Adjusted p
Acute kidney injury (no. (%))
200 (21.3)
46 (14.6)
54 (17.2)
100 (32.3)
<0.0001
Renal replacement therapy (no. (%))
213 (22.2)
17 (5.3)
54 (16.9)
142 (44.7)
<0.0001
Length of stay in ICU – days (mean±SD)
15.4 ± 16.5
12.2 ± 14.1
16.3 ± 16.1
17.8 ± 18.5
0.0001
Length of stay in hospital – days (mean±SD)
29.1 ± 27.8
26.2 ± 24.1
30.6 ± 29.3
30.5 ± 29.7
0.04
Duration of mechanical ventilation – days (median [Q1-Q3])
7 [2-15]
4 [1-10]
7 [3-17]
10 [3-18]
<0.0001
Time to suspension of vasopressor or inotropic agents– days (median [Q1-Q3])
3 [2-7]
2 [1-5]
3 [2-8]
5 [2-9]
0.006
Clinical resolution of the primary infection – (no. (%))
488 (61.4)
190 (72.8)
169 (63.30)
129 (48.3)
0.001
Duration of the primary infection – days (median [Q1-Q3])
8 [4-13]
6.5 [4-10]
9 [4-15]
9 [5-15]
0.0006
Incidence of secondary acquired infection during the study (no. (%))
246 (25.7)
61 (19.1)
89 (27.8)
96 (30.2)
0.10
The relations between tertiary outcomes and presepsin concentration were analyzed with a multivariate logistic regression model and a linear regression model for categorical and
continuous variables, respectively. The covariates included in the model were age, immunodeficiency, shock, mechanical ventilation and SOFA score at baseline.
7
Supplementary Table 4: Prognostic discrimination and reclassification of presepsin at day 1
ICU mortality
Patients
Model
AUC [95%CI]
p
IDI [95%CI]
Continuous
NRI
p
Clinical model
All
Shock
No
shock
Events
correctly
reclassified
Non-events
correctly
reclassified
20%
34%
0.76 [0.74-0.80]
0.80 [0.77-0.83]
Clinical model
0.74 [0.69-0.78]
0.80 [0.76-0.84]
Clinical model
0.75 [0.69-0.80]
IDI [95%CI]
Continuous
NRI
p value
Events
correctly
reclassified
Non-events
correctly
reclassified
0.04 [0.03-0.06]
p<0.0001
0.55 [0.41-0.69]
p<0.0001
0.09 [0.06-0.11]
p<0.0001
0.61 [0.43-0.79]
p<0.0001
0.03 [0.01-0.05]
p=0.01
0.39 [0.16-0.62]
p=0.0009
0.001
0.04 [0.03-0.05]
p<0.0001
0.51 [0.38-0.63]
p<0.0001
18%
33%
0.002
0.06 [0.04-0.08]
p<0.0001
0.58 [0.42-0.75]
p<0.0001
21%
38%
0.39
0.003 [-0.0040.01] p=0.36
0.21 [0.00-0.41]
p=0.05
5%
15%
0.81 [0.78-0.84]
23%
38%
0.81 [0.78-0.85]
0.81 [0.77-0.85]
0.17
0.77 [0.72-0.82]
p
0.78 [0.74-0.82]
<0.0001
Clinical model
+ presepsin
AUC [95%CI]
0.79 [0.76-0.81]
0.002
Clinical model
+ presepsin
Clinical model
+ presepsin
90-day mortality
15%
24%
0.81 [0.77-0.86]
The variables included in the clinical model are listed in the legend of Table 3. AUC: area under the curve; IDI: integrated discrimination improvement; continuous NRI:
category-free net reclassification improvement. Discrimination was evaluated by C-index comparing the clinical model with the same model plus day-1 concentration of
presepsin with the DeLong test.
8
Supplementary Table 5: Interaction between presepsin at day 1 and albumin on outcome
ICU mortality
(No. events/No. patients (%))
90 day mortality
(No. events/No. patients (%))
Group
All
Albumin
Crystalloids
P
All
Albumin
Crystalloids
P
Presepsin < 946 ng/L
68/479 (14.2)
39/242 (16.1)
29/237 (12.2)
0.22
122/473 (25.8)
67/238 (28.2)
55/235 (23.4)
0.24
Presepsin ≥ 946 ng/L
185/479 (38.6)
89/232 (38.4)
96/247 (38.9)
0.91
247/472 (52.3)
118/229 (51.5)
129/243 (53.1)
0.73
Breslow-Day for interaction between presepsin on day 1 and randomized treatment: p= 0.29 for ICU mortality, p=0.27 for 90-day mortality.
9
Supplementary Table 6: Prognostic value of changes in presepsin concentration over time
Outcome
Patients
Changes from day 1 to day 2
No. events / no.
OR [95%CI]
Changes from day 1 to day 7
p
patients (%)
ICU mortality
90-day mortality
No. events / no.
OR [95%CI]
p
146/716 (20.4)
2.74 [2.06-3.65]
<0.0001
64/327 /19.6)
3.46 [2.17-5.50]
<0.0001
82/389 (21.1)
2.39 [1.64-3.49]
<0.0001
patients (%)
All
216/870 (24.8)
2.43 [1.62-3.65]
<0.0001
Severe sepsis
84/384 (21.9)
3.28 [1.74-6.19]
0.0002
Shock
132/486 (27.2)
2.07 [1.25-3.44]
0.005
All
325/858 (37.9)
1.59 [1.11-2.28]
0.01
242/704 (24.4)
1.90 [1.49-2.42]
<0.0001
Severe sepsis
123/375 (43.7)
1.93 [1.09-3.43]
0.02
98/318 (30.8)
1.71 [1.20-2.44]
0.003
Shock
202/483 (41.8)
1.43 [0.91-2.26]
0.12
144/386 (37.3)
1.80 [1.31-2.47]
0.0003
The results of the logistic models for the association between changes in presepsin concentration over time (after log-transformation) and mortality are shown as odd ratio and
95% confidence interval. The covariates, as listed in the legend of Table 3, were introduced in the multivariable logistic models if they had a p<0.20 at univariate analyses and
selected in the final model after backward elimination.
10
Supplementary Figure 1: Baseline presepsin and disease severity
Baseline presepsin concentrations according to the SOFA score (A) or the numbers of organ dysfunction or failure at
randomization (B). Number of patients with organ-specific failure on day 1 (SOFA scores 3-4): respiration (335, 35.9%
of those with organ-specific evaluation of SOFA score), coagulation (124, 13.1%), liver (30, 3.2%), cardiovascular
(578, 61.0%), kidney (204, 21.6%). P across categories by Kruskal-Wallis test.
11
Supplementary Figure 2: Time course of plasma presepsin levels according to microorganism culture results and
appropriateness of antibiotic therapy
Median [Q1-Q3] concentrations of presepsin at different times according to microorganism culture results in blood (a)
or at the primary site of infection (b) and the appropriateness of empirical antibiotic therapy, as defined in the Methods.
The number of patients in each category is shown in the columns. Two-way analyses for repeated measures adjusted for
SOFA score at baseline. Time course by blood culture: time (p=0.05), appropriateness of antibiotic therapy (p=0.93),
interaction time x appropriateness of antibiotic therapy (p=0.0009). Time course by site culture: time (p=0.57),
appropriateness of antibiotic therapy (p=0.53), interaction time x appropriateness of antibiotic therapy (p=0.002).
12
Supplementary Figure 3: Time course of presepsin according to ICU survival
Concentrations of presepsin are shown as median [Q1-Q3]. The number of patients is shown above the columns.
13
Supplementary Figure 4: Presepsin concentration on day 1 and mortality
Kaplan–Meier survival curves for ICU or 90-day mortality by tertiles of presepsin concentration measured at day 1. The
number of patients at risk is shown below the curves. P for log-rank test.
14