Original Article
doi: 10.1111/joim.12054
Soluble urokinase plasminogen activator receptor is
compartmentally regulated in decompensated cirrhosis and
indicates immune activation and short-term mortality
H. W. Zimmermann1,2, P. A. Reuken3,4, A. Koch1, M. Bartneck1, D. H. Adams2, C. Trautwein1, A. Stallmach3,4,
F. Tacke1 & T. Bruns2,3,4
From the 1Department of Medicine III, University Hospital Aachen, Aachen, Germany; 2NIHR Biomedical Research Unit and Centre for Liver
Research, School of Immunity and Infection, University of Birmingham, Birmingham, UK; 3Department of Internal Medicine IV, Jena
University Hospital, Friedrich Schiller University Jena, and 4Integrated Research and Treatment Center – Center for Sepsis Control and Care
(CSCC), Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany
Abstract. Zimmermann HW, Reuken PA, Koch A,
Bartneck M, Adams DH, Trautwein C, Stallmach A,
Tacke F, Bruns T (University Hospital Aachen,
Aachen, Germany; University of Birmingham,
Birmingham, UK; Jena University Hospital,
Friedrich Schiller University Jena, Jena, Germany;
Integrated Research and Treatment Center – Center
for Sepsis Control and Care (CSCC), Jena University
Hospital, Friedrich Schiller University Jena,
Jena, Germany). Soluble urokinase plasminogen
activator receptor is compartmentally regulated in
decompensated cirrhosis and indicates immune
activation and short-term mortality. J Intern Med
2013; 274: 86–100.
Objective. Patients with decompensated cirrhosis are
susceptible to bacterial infections, which are associated with organ failure and a high mortality rate.
Reliable biomarkers are needed to identify patients
who require intensified treatment. Our objective
was to study the regulation and prognostic relevance of elevated concentrations of soluble urokinase plasminogen activator receptor (suPAR) in
patients with advanced cirrhosis.
Design, setting and participants. We examined the associations between serum and ascitic fluid (AF) suPAR
and liver function, bacterial infection, and shortterm mortality in 162 consecutive patients with
decompensated cirrhosis undergoing diagnostic
paracentesis in a tertiary health care centre in
Germany.
Main outcome measure. Twenty-eight-day mortality.
Results. Circulating suPAR levels were increased in
patients with decompensated cirrhosis and correF. Tacke and T. Bruns contributed equally.
86
ª 2013 The Association for the Publication of the Journal of Internal Medicine
lated with the severity of liver dysfunction and
systemic inflammation but were not indicative of
bacterial infection. Circulating suPAR levels
>14.4 ng mL 1 predicted 28-day mortality, even
after adjustment for liver function and confounders
[HR = 3.05 (1.35–6.90); P = 0.0076] equal to the
MELD score (AUC = 0.71; 95% CI = 0.61–0.81;
P < 0.001). Cut-off levels derived from cohorts
without liver disease were not applicable due to
the low specificity. AF suPAR levels were elevated
during spontaneous bacterial peritonitis (SBP), but
not during episodes in which bacteria or bacterial
DNA was translocated into the ascites. AF suPAR
levels correlated poorly with systemic suPAR but
were associated with a more severe course of SBP
and a worse outcome. In vitro experiments revealed
that monocytes, and to a lesser extent neutrophils,
secrete suPAR after Toll-like-receptor ligation,
which led to rapid urokinase plasminogen activator
receptor cleavage followed by increased synthesis.
Conclusion. Blood and ascitic suPAR levels provide
distinct, but relevant prognostic information on the
severity of complications in patients with end-stage
liver disease.
Keywords: ascites, biomarker, cirrhosis, spontaneous bacterial peritonitis, survival.
Abbreviations: AF, ascitic fluid; AKI, acute kidney
injury; ALT, alanine aminotransferase; bactDNA,
bacterial deoxyribonucleic acid; CD, cluster of differentiation; CI, confidence interval; CLD, chronic
liver disease; CRP, C-reactive protein; E. coli, Escherichia coli; ELISA, enzyme-linked immunosorbent
assay; G-PLD, phosphatidylinositol-glycan-specific
phospholipase D; HR, hazard ratio; INR, international normalized ratio; LBP, lipopolysaccharidebinding protein; LPS, lipopolysaccharide; LTX, liver
H. W. Zimmermann et al.
transplantation; MELD, model for end-stage liver
disease; PCR, polymerase chain reaction; ROC,
receiver-operating characteristic; SAAG, serumascites albumin gradient; SBP, spontaneous bacterial peritonitis; SIRS, systemic inflammatory
response syndrome; SOFA, sequential organ failure
Introduction
Decompensated cirrhosis is associated with a 1year cumulative mortality between 28% and 52%
(median = 39%) due to liver failure and the complications of liver failure [1]. Bacterial infections,
including spontaneous bacterial peritonitis (SBP),
pneumonia, urinary tract infections, and bacteraemia, occur in one-third of hospitalized patients
with decompensated cirrhosis and contribute to
increased mortality by triggering sepsis and organ
failure [2–5]. One-year mortality rates in patients
with decompensated cirrhosis and bacterial infections exceed 60% and have changed little over the
last three decades [2]. Reliable biomarkers that
identify patients with a high risk of mortality would
be invaluable in deciding when and in which
patients to escalate medical care, and in selecting
patients for liver transplantation.
The model for end-stage liver disease (MELD) score
has proven to be an accurate predictor of shortand long-term survival in hospitalized and ambulatory patients with cirrhosis [6]. Although organ
dysfunction scores, such as the Sequential Organ
Failure Assessment (SOFA), outperform MELD in
predicting short-term mortality in critically ill
patients with cirrhosis, organ dysfunction scores
are rarely used outside the ICU setting [7]. Clinical
and laboratory criteria, such as the presence of
systemic inflammatory response syndrome (SIRS)
and elevated C-reactive protein (CRP) levels, have
been reported to predict mortality in patients with
cirrhosis because of the association with bacterial
infections and endotoxaemia [8–10]; however, the
use of composite scores or clinical data is susceptible to confounders, which may limit applicability
in clinical practice.
The soluble urokinase plasminogen activator
receptor (suPAR) is a promising prognostic biomarker. There is evidence that elevated levels of serum
suPAR are associated with an unfavourable outcome in patients with sepsis [11–13] and bacteraemia [14–16], patients admitted to the emergency
department with suspected bacterial infections
suPAR in advanced cirrhosis
assessment; spp., species; suPAR, soluble urokinase plasminogen activator receptor; TLR, Toll-like
receptor; TNF-a, tumour necrosis factor-alpha;
uPAR, urokinase plasminogen activator receptor
(CD87); WBC, white blood cells.
[17] and patients with human immunodeficiency
virus-1 (HIV-1) infections [18]. Soluble urokinase
plasminogen activator receptor is released into
body fluids after enzymatic removal or proteolytic
cleavage of cell surface CD87 (uPAR) from various
immunologically active cells, acting as a chemotactic agent and as a scavenger receptor for uPA
and vitronectin [19]. Despite the role of suPAR as
an emerging biomarker of immune activation in
infectious disease and low-grade inflammation
[20], the origin and regulation of suPAR is not fully
understood [21, 22].
Systemic suPAR levels correlate with liver fibrosis
and inflammation in patients with chronic liver
diseases in the absence of systemic inflammation
and overt bacterial infection [23–25]. Furthermore,
the highest circulating suPAR levels in acutely ill
medical patients are detected in patients with
impaired liver function and chronic liver disease
[12, 17, 26]. The prognostic significance of elevated
levels of suPAR in end-stage cirrhotic patients who
are at risk of infectious complications, however,
has not been evaluated.
Thus, we determined the levels of suPAR in
matched ascitic fluid (AF) and blood as an indicator
of short-term mortality in a high-risk cohort of
patients with decompensated cirrhosis and suspected infection. We investigated the cellular
expression and release of uPAR in neutrophils
and monocytes in vitro in response to Toll-like
receptor (TLR) ligands present during bacterial
infections and endotoxaemia.
Methods
Study design
One hundred sixty-two consecutive patients with
decompensated liver cirrhosis admitted to the Jena
University Hospital between September 2010 and
April 2012 were prospectively recruited for the
determination of suPAR serum and AF concentrations. The inclusion criteria were as follows: cirrhosis defined by clinical, laboratory, or histological
criteria; the presence of ascites accessible to
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
87
H. W. Zimmermann et al.
suPAR in advanced cirrhosis
diagnostic abdominal paracentesis; and 18 years
of age and suspected bacterial infection. The exclusion criteria included acute alcoholic hepatitis,
peritoneal carcinomatosis, secondary peritonitis,
acute pancreatitis, and tuberculous peritonitis.
The primary outcome of the study was the prediction
of 28-day mortality by elevated concentrations of
serum suPAR. The secondary outcomes included
the association of serum suPAR with 90-day mortality and bacterial infections, the association of AF
suPAR with bacterial translocation, SBP, and SBPrelated complications and identification of cellular
suPAR sources using in vitro models of inflammation and infection.
positive AF culture (bacterascites), a urine WBC
>20 lL 1 with a positive bacterial culture >105
colony-forming units (urinary tract infection), biliary obstruction with a positive bile culture (acute
cholangitis), or new infiltrates on chest x-ray with a
positive quantitative culture from a bronchoalveolar lavage (pneumonia). Bacterial infections that
were diagnosed using a combination of clinical and
imaging criteria and did not fulfil the criteria above
were categorized as clinical infections. The survival
and transplantation status were assessed in July
2012 by reviewing electronic medical records and
death certificate data, or by interviewing the
appropriate general practitioner.
Written informed consent was obtained from all of
the patients, and the study protocol conformed to
the ethical guidelines of the 1975 Declaration of
Helsinki and was approved by the local Ethics
Committee (26.08.2010; no. 2880-08/10).
Blood samples for the control group with compensated cirrhosis without ascites were obtained from
patients recruited at the University Hospital of
Aachen [27].
Ex vivo and in vitro analyses
Samples and patient data
At the time of enrolment, blood pressure, heart
rate, body temperature, and samples of blood and
AF were obtained. The AF total cell count was
determined using an automated XE blood cell
counter (Sysmex, Norderstedt, Germany). In samples with a cell count 250 mm ³, the polymorphonuclear (PMN) count was manually determined
using a counting chamber. Bacterial cultures were
performed in BacT/ALERT blood culture bottles
(Biom
erieux, Durham, NC, USA) at 37 °C after
inoculation with at least 10 mL of AF or blood.
Protein and albumin concentrations, blood count,
liver function tests, serum creatinine and sodium
levels were determined by routine laboratory
analysis.
The following variables were collected at study
inclusion: age; gender; aetiology of cirrhosis; comorbidities; medication; SIRS criteria within the
last 24 h; evidence of bacterial infection within the
last 7 days (clinical, microbiologic and imaging
criteria); increased serum creatinine (>0.5 mg dL 1)
or bilirubin (>2 mg dL 1) over baseline (hospitalization or within 30 days); and MELD and ChildPugh scores. Proven bacterial infections were
defined as microbiologically diagnosed infections,
including, positive blood cultures (spontaneous
and secondary bacteraemia), an AF neutrophil
count 250 lL 1 with or without a positive AF
culture (culture-negative and culture-positive
SBP), an AF neutrophil count <250 lL 1 with a
88
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
In patients with culture-negative, non-neutrocytic
ascites, AF was assessed for microbial DNA fragments (bactDNA) as an indicator of bacterial
translocation [28] using the VYOO multiplex polymerase chain reaction (PCR) system (SIRS-Lab,
Jena, Germany), as previously described [29].
Soluble uPAR, lipopolysaccharide-binding protein
(LBP), interleukin (IL)-10, IL-6 and tumour necrosis factor-alpha (TNF-a) were measured by ELISA,
as described previously [24, 30]. Monocytes and
neutrophils were isolated from blood obtained from
healthy volunteers and stimulated in vitro using
recombinant human TNF-a (Peprotech, London,
UK) and TLR agonists. The surface expression of
CD87 was determined using flow cytometry [Cyan;
Beckman Coulter, High Wycombe, UK (for details,
see Data S1)].
Statistical analysis
Patient data are reported as the median/range for
continuous variables or number/fraction for discrete data. The statistical differences between
groups were analysed using a non-parametric
Mann–Whitney U-test or Kruskal–Wallis test with
Dunn’s post hoc test for continuous data or Fisher’s exact test for discrete data, respectively, as
two-sided tests. Nonparametric measurement of
statistical dependence between two variables was
performed by calculating the Spearman’s rank
correlation coefficient [rho (rs)]. Ninety-day survival
was assessed using the Kaplan–Meier method,
H. W. Zimmermann et al.
suPAR in advanced cirrhosis
Table 1 Serum suPAR levels stratified for demographic and
clinical baseline characteristics in patients with advanced
cirrhosis
Table 1 (Continued )
Serum suPAR
Median
Serum suPAR
Median
No (%)
(interquartile
(n = 162)
range)
P-value
Gender
Female
Male
42 (26)
12.0 (9.3–17.2)
120 (74)
12.4 (9.1–17.3)
123 (76)
12.4 (9.5–18.1)
39 (24)
10.5 (7.7–15.9)
0.98
Nonalcoholic
0.09
86 (53)
11.0 (9.1–15.7)
No
76 (47)
13.8 (9.3–19.6)
0.06
B
46 (28)
9.9 (6.8–13.9)
116 (72)
13.6 (9.9–19.2)
0.001
Yes
Unknown
140 (86)
11.8 (8.9–17.1)
13 (8)
13.7 (10.5–16.3)
9 (6)
14.5 (10.2–38.5)
0.34
49 (30)
13.0 (9.0–17.9)
Yes
94 (58)
11.2 (9.3–16.5)
Unknown
19 (12)
13.5 (9.0–30.8)
Yes
147 (91)
12.0 (9.1–17–4)
15 (9)
13.7 (9.9–17.1)
0.41
123 (76)
12.4 (9.4–17.5)
Hepatocellular
24 (15)
12.0 (9.3–16.7)
15 (9)
9.4 (6.8–16.8)
106 (65)
12.1 (9.1–17.7)
56 (35)
12.3 (9.2–16.7)
0.91
Yes
0.27
72 (44)
10.8 (9.1–16.2)
Yes
90 (56)
12.8 (9.3–19.6)
0.97
0.20
Bacterial infection
No
67 (41)
12.4 (9.1–16.8)
Clinical
39 (24)
12.4 (8.2–17.5)
Proven
56 (35)
11.4 (9.2–18.1)
Urinary tract
7 (4)
15.3 (10.6–21.6)
infection
30 (19)
10.0 (8.6–14.4)
140 (86)
11.9 (8.9–17.0)
22 (14)
14.3 (9.6–22.7)
Blood culture result
Negative
0.32
Ascitic fluid culture result
Negative
141 (87)
21 (13)
12.2 (9.1–17.0)
0.27
14.7 (10.0–30.7)
Dialysisa
No
153 (94)
12.2 (9.1–17.5)
9 (6)
11.3 (9.2–16.7)
No
72 (44)
11.6 (9.0–16.7)
Yes
90 (56)
12.7 (9.4–18.2)
144 (89)
12.1 (9.1–16.9)
10 (6)
11.1 (9.9–25.9)
8 (5)
16.4 (8.0–23.7)
Terlipressin
Other
0.77
0.44
0.65
Albumin replacementa
No
Yes
Systemic inflammatory response syndrome
No
16.1 (8.6–25.3)
vasopressors
Diabetes mellitus
No
21 (13)
bacterascites
No
carcinoma
Other
14.7 (9.8–19.4)
Vasoactive therapya
Malignant tumour
No
12.4 (9.1–16.8)
37 (23)
Antibiotic therapya
Gastrointestinal haemorrhagea
No
67 (41)
SBP, including
Yes
Oesophageal or gastric fundal varices
No
0.27
No
Positive
Portal vein thrombosis
No
P-value
Positive
Child-Pugh class
C
range)
Bacterial infection
Other
Alcohol abstinence
Yes
(interquartile
(n = 162)
Pneumonia
Aetiology
Alcoholic
No (%)
0.95
102 (63)
11.8 (8.7–16.9)
60 (37)
12.7 (9.8–20.6)
0.17
P-values derived from nonparametric testing (Mann–
Whitney U-test or Kruskal–Wallis test).
a
Within the last 14 days prior to inclusion.
pairwise log-rank test, and univariate and
multivariate Cox proportional hazards models, as
indicated. Patients were censored at the completion of short-term follow-up or on the day of liver
transplantation. In vitro data are reported as the
mean and standard error of the mean, and tested
for significance using a paired t-test or repeated
measures ANOVA with Dunnet’s post hoc test.
Statistical calculations were performed using SPSS
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
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H. W. Zimmermann et al.
suPAR in advanced cirrhosis
18 (SPSS, Inc., Chicago, IL, USA) and GRAPHPAD
PRISM 5 (La Jolla, CA, USA).
Results
Elevated levels of serum suPAR correlate with organ function and
inflammation, but not with bacterial infection and SIRS
Of the 162 patients with decompensated cirrhosis in
the study population, 120 were men, 24–91 years of
age (median = 58 years), and the majority presented with alcoholic liver disease (Table 1). Even
in the absence of overt bacterial infection and SIRS,
patients with cirrhosis and ascites had significantly
higher serum suPAR levels (median = 12.9 ng mL 1;
(a)
(d)
range = 5.9–45.7 ng mL 1; n = 32) than an age- and
gender-matched control group of patients with
compensated cirrhosis without ascites (median
= 8.9 ng mL 1; range = 2.5–20.0 ng mL 1; n = 39;
P < 0.001; Fig. 1a). Surprisingly, in patients with
decompensated cirrhosis the serum suPAR concentrations did not significantly differ with respect to
the presence of SIRS or underlying bacterial infection (Table 1; Fig. 1b). In contrast, circulating suPAR correlated with the MELD (rs = 0.44; P < 0.001)
and Child-Pugh scores (rs = 0.38; P < 0.001;
Fig. 1c), and with the composites of those scores
(serum bilirubin, albumin, creatinine, and INR),
white blood cell count, CRP, and serum sodium
(c)
(b)
(e)
(f)
(g)
Fig. 1 Circulating suPAR levels in advanced cirrhosis. (a) Even in the absence of bacterial infection and systemic
inflammatory response syndrome (SIRS), suPAR serum concentrations were higher in patients with cirrhosis and ascites
(n = 32) than in patients with compensated cirrhosis (n = 39). Tukey’s box plots are shown and P values from Mann–
Whitney U-test are indicated. (b) SuPAR serum concentration in 162 patients with decompensated cirrhosis showed no
significant differences with respect to the presence of SIRS and clinically diagnosed or microbiologically proven infection.
Distribution and median are shown and P value from Kruskal–Wallis test is indicated (for frequencies of bacterial infections,
see Table 1). (c) Circulating suPAR correlated (nonparametric correlation) with the MELD and Child-Pugh scores in patients
with cirrhosis and ascites. Spearman’s rank correlation coefficients are indicated. (d) Serum suPAR levels in patients who
died within 28 days after inclusion were higher than patients who survived or underwent liver transplantation (LTX).
Median and P values from the Mann–Whitney U-test are indicated. (e) Receiver operating characteristic curve showing the
optimum cut-off (14.4 ng mL 1) to predict 28-day mortality in this cohort of patients with advanced cirrhosis and suspected
infection, as well as proposed cut-offs for mortality in other cohorts between 6.4 and 9.0 ng mL 1 [12, 17, 24]. (f) Kaplan–
Meier analysis of survival demonstrating higher 90-day mortality in patients with serum suPAR levels 14.4 ng mL 1
(n = 61) than patients with lower AF serum levels (n = 101). P value from log-rank test and censored cases due to liver
transplantation are indicated. (g) Hazard ratios and 95% confidence intervals for elevated serum suPAR levels
14.4 ng mL 1 to predict death within 28 days are indicated for the overall cohort and for subgroups with respect to
aetiology of cirrhosis or the presence of bacterial infections.
90
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
H. W. Zimmermann et al.
suPAR in advanced cirrhosis
Table 2 Nonparametric correlation of serum and ascitic suPAR levels with markers of liver function and inflammation
Serum suPAR
Ascitic suPAR
All patients (n = 162)
No SBP (n = 132)
SBP (n = 30)
MELD score
0.44 (P < 0.001)
0.01 (P = 0.87)
0.30 (P = 0.11)
Serum MR-proADM
0.42 (P < 0.001)
0.19 (P = 0.04)
0.40 (P = 0.04)
Bilirubin
0.38 (P < 0.001)
0.04 (P = 0.66)
0.11 (P = 0.55)
Child-Pugh score
0.38 (P < 0.001)
0.15 (P = 0.08)
0.15 (P = 0.42)
INR
0.33 (P < 0.001)
0.15 (P = 0.08)
0.29 (P = 0.13)
WBC
0.32 (P < 0.001)
0.13 (P = 0.13)
0.44 (P = 0.02)
C-reactive protein
0.26 (P = 0.001)
0.08 (P = 0.379)
0.34 (P = 0.06)
Creatinine
0.25 (P = 0.002)
0.22 (P = 0.01)
0.31 (P = 0.10)
Serum interleukin-10
0.22 (P = 0.006)
0.10 (P = 0.28)
0.03 (P = 0.90)
Serum LBP
0.14 (P = 0.07)
0.14 (P = 0.12)
0.40 (P = 0.03)
Heart rate
0.13 (P = 0.10)
0.07 (P = 0.46)
0.23 (P = 0.23)
ALT
0.09 (P = 0.26)
0.18 (P = 0.04)
0.26 (P = 0.17)
Serum TNF-a
0.03 (P = 0.67)
0.13 (P = 0.15)
0.21 (P = 0.28)
Temperature
0.01 (P = 0.91)
0.07 (P = 0.40)
0.06 (P = 0.77)
Mean arterial pressure
0.00 (P = 0.97)
0.03 (P = 0.76)
0.06 (P = 0.75)
Serum interleukin-6
0.01 (P = 0.93)
0.01 (P = 0.88)
0.11 (P = 0.55)
Platelets
0.03 (P = 0.70)
0.34 (P < 0.001)
0.24 (P = 0.20)
SAAG
0.05 (P = 0.58)
0.19 (P = 0.03)
0.20 (P = 0.30)
Ascitic WBC count
0.09 (P = 0.28)
0.09 (P = 0.31)
0.02 (P = 0.91)
Age
0.17 (P = 0.03)
0.02 (P = 0.83)
0.15 (P = 0.45)
Ascitic protein
0.20 (P = 0.01)
0.43 (P < 0.001)
0.06 (P = 0.75)
Sodium
0.20 (P = 0.01)
0.03 (P = 0.73)
0.19 (P = 0.33)
Serum albumin
0.30 (P < 0.001)
0.17 (P = 0.05)
0.21 (P = 0.26)
Spearman’s rank correlation coefficient and significance level in nonparametric correlation are indicated. MELD, model
for end-stage liver disease; MR-proADM: mid-regional pro-adrenomedullin; WBC: white blood cell count; INR:
international normalized ratio; LBP: lipopolysaccharide-binding protein; ALT: alanine aminotransferase; SAAG: serumascites albumin gradient.
(Table 2). There were no significant correlations
between serum suPAR and heart rate, mean
arterial pressure, serum IL-6, or serum TNF-a.
Serum suPAR identifies patients at risk for short-term mortality
independent of MELD score, bacterial infection, and level of
inflammation
Twenty-eight days after inclusion, 34 patients
(21%) had died, six (4%) underwent liver transplantation, and 122 (75%) were alive without
transplantation. The documented causes of death
included infection/sepsis (n = 16), renal failure
and hepato-renal syndrome (n = 7), acute-onchronic liver failure and multi-organ failure
(n = 7), gastrointestinal haemorrhage (n = 2), and
other causes (n = 2). Nonsurvivors presented with
significantly higher levels of serum suPAR at
inclusion than survivors or patients who subsequently underwent transplantation (Fig. 1d). The
receiver operating characteristics (ROC) curve suggested a serum suPAR concentration of
14.4 ng mL 1 as the best cut-off for predicting
28-day mortality with a sensitivity of 71% and a
specificity of 71%, whereas other proposed cut-offs
(6.4–9.0 ng mL 1) [12, 17, 24] had poor discrimination of survival in this cohort (Fig. 1e). The areas
under the ROC curve (AUROC) for predicting death
within 28 days were 0.71 (95% CI = 0.61–0.81;
P < 0.001) for serum suPAR, 0.71 (95% CI = 0.59–0.82;
P < 0.001) for the MELD score, and 0.66 (95%
CI = 0.56–0.79; P = 0.004) for CRP.
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
91
H. W. Zimmermann et al.
suPAR in advanced cirrhosis
Table 3 Short-term survival
Follow-up at 28 days
Characteristics
Multivariate modelb
Univariate model
No (%) who
No (%) alivea
Hazard ratio
died (n = 34)
(n = 128)
(95% CI)
P-value
Adjusted hazard
ratio (95% CI)
P-value
0.84
–
–
0.44 (0.18c)
–
–
0.18
–
–
0.01 (<0.001c)
Removed from model
n.s.
0.001 (<0.001c)
1.06 (1.01–1.11) per
0.01c
(A) Risk factors for 28-days mortality
Gender
Female
9 (27)
33 (26)
1.00 (ref)
25 (74)
95 (74)
0.92 (0.43–1.98)
<55 years
11 (32)
40 (31)
1.00 (ref)
55–64 years
10 (29)
52 (41)
0.74 (0.32–1.75)
65 years
13 (38)
36 (28)
1.27 (0.57–2.84)
Alcoholic
23 (67)
100 (78)
Non-alcoholic
11 (32)
28 (22)
1.63 (0.80–3.35)
3 (9)
43 (34)
1.00 (ref)
31 (91)
85 (66)
4.63 (1.41–15.14)
<15
8 (24)
54 (42)
1.00 (ref)
15–19
4 (12)
24 (19)
1.08 (0.33–3.58)
Male
Age
Aetiology
1.00 (ref)
Child-Pugh score
<10
10
MELD score
1-point increase
20–24
25
6 (18)
30 (23)
1.30 (0.45–3.74)
16 (47)
20 (16)
4.54 (1.94–10.62)
Malignant tumour
No
24 (71)
99 (77)
1.00 (ref)
HCC
5 (15)
19 (15)
1.07 (0.41–2.81)
Other
5 (15)
10 (8)
1.77 (0.69–4.65)
No
22 (65)
84 (66)
1.00 (ref)
Yes
12 (35)
44 (34)
1.00 (0.50–2.03)
0.51
–
–
0.99
–
–
0.05
–
–
0.004
1.00 (ref)
0.04
Diabetes mellitus
SIRS
No
10 (29)
62 (48)
1.00 (ref)
Yes
24 (71)
66 (52)
2.09 (1.00–4.37)
6 (18)
61 (48)
1.00 (ref)
Bacterial infection
No
Clinical
Proven
8 (24)
31 (24)
2.31 (0.80–6.66)
1.97 (0.68–5.70)
20 (59)
36 (28)
4.56 (1.83–11.35)
3.23 (1.28–8.14)
31 (91)
123 (96)
3 (9)
5 (4)
31 (91)
116 (91)
3 (9)
12 (9)
SBP in history
No
Yes
1.00 (ref)
0.24
–
–
–
–
2.03 (0.62–6.66)
GI haemorrhage
No
Yes
92
1.00 (ref)
1.00 (0.31–3.27)
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
1
H. W. Zimmermann et al.
suPAR in advanced cirrhosis
Table 3 (Continued )
Follow-up at 28 days
Characteristics
Multivariate modelb
Univariate model
No (%) who
No (%) alivea
Hazard ratio
died (n = 34)
(n = 128)
(95% CI)
P-value
Adjusted hazard
ratio (95% CI)
P-value
0.12
–
–
0.003 (0.003c)
Removed from model
n.s.
1.00 (ref)
0.009
Beta-blocker
No
22 (65)
62 (48)
1.00 (ref)
Yes
12 (35)
66 (52)
0.57 (0.28–1.16)
7 (21)
65 (51)
1.00 (ref)
27 (79)
63 (49)
3.49 (1.52–8.02)
10 (29)
91 (71)
1.00 (ref)
24 (71)
37 (29)
4.83 (2.31–10.12)
C-reactive proteind
<29 mg L
29 mg L
1
1
Serum suPAR
<14.4 ng mL
1
14.4 ng mL
1
<0.001 (0.002c)
2.93 (1.31–6.55)
Risk factors for 28-day mortality adjusted for
Risk factors for 90-day mortality adjusted for age
age and cancer
and cancer
Adjusted hazard ratio (95% CI)
P-value
Adjusted hazard ratio (95% CI)
P value
<0.001c
(B) Prognostic relevance of serum suPAR for 28- and 90-day mortality
1.06 (1.02–1.12) per 1-point increase
0.008c
1.07 (1.03–1.11) per 1-point increase
No
1.00 (ref)
0.06
1.00 (ref)
Clinical
2.13 (0.73–6.23)
2.09 (0.97–4.47)
Proven
3.06 (1.20–7.79)
2.51 (1.30–4.83)
MELD score
Bacterial infection
0.02
Serum suPAR
<14.4 ng mL
14.4 ng mL
1
1.00 (ref)
1
3.05 (1.35–6.90)
0.007
1.00 (ref)
0.03
1.94 (1.09–3.46)
MELD, model for end-stage liver disease; SIRS, systemic inflammatory response syndrome, SBP, spontaneous bacterial
peritonitis; GI, gastrointestinal; suPAR, soluble urokinase plasminogen activator receptor.
a
Patients who underwent liver transplant within 28 days were censored at the date of transplantation.
b
Multivariate model includes significant variables from univariate regression in a stepwise backward analysis (P < 0.05).
c
P-value in Cox hazard regression, when treated as a continuous variable.
d
Cut-off derived from Cervoni et al. [10].
Using the derived cut-off, elevated levels of suPAR
were associated with a HR of 4.83 (95% CI = 2.31–
10.12) for 28-day mortality (P < 0.001; Table 3A,
Fig. 1f) in patients with and without bacterial
infections (Fig. 1g). Other parameters associated
with increased 28-day mortality in univariate
analysis were a higher MELD score, bacterial
infection, and elevated CRP (Table 3A). Serum
suPAR levels >14.4 ng mL 1 remained a significant
predictor of 28-day mortality, with a HR of 2.93
(95% CI = 1.31–6.55; P = 0.009) in multivariate
Cox regression after adjustment for covariates
(MELD score and bacterial infection).
Elevated levels of circulating suPAR were predictive
of survival, even after 90 days of follow-up in
univariate (HR = 2.93; 95% CI = 1.73–4.96; P <
0.001) and multivariate analyses (HR = 1.94; 95%
CI = 1.09–3.46; P = 0.03; Table 3B).
Ascitic fluid concentrations of suPAR indicate SBP and correlate with
severity
Serum and AF concentrations of suPAR were
weakly correlated (r = 0.16; P = 0.05), but showed
an interesting pattern of differential regulation in
which subgroups of patients predominantly
showed AF or serum elevation (Fig. 2a).
Patients with SBP had higher concentrations of AF
suPAR
(median = 12.8 ng mL 1;
range = 3.6–
74.9 ng mL 1) than patients without SBP
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
93
H. W. Zimmermann et al.
suPAR in advanced cirrhosis
(median = 11.1 ng mL 1; range = 3.6–74.9; P =
0.025; Fig. 2b) and correlated with the concentration of AF lipopolysaccharide-binding protein (LBP)
(rs = 0.54; P = 0.002; Fig. 2c). The AF suPAR concentration suggested SBP at an AUROC of 0.70 (95%
(median = 9.4 ng mL 1; range = 2.2–27.7 ng mL 1;
P = 0.001). Furthermore, the levels of AF suPAR were
higher in patients with culture-positive SBP
(median = 17.8 ng mL 1; range = 8.0–60.9 ng
mL 1) than patients with culture-negative SBP
(a)
(b)
(e)
(c)
(d)
(f)
Fig. 2 Ascitic fluid suPAR levels. (a) Serum and ascitic fluid (AF) concentrations of suPAR did not correlate well and
revealed the highest AF concentrations in patients with spontaneous bacterial peritonitis (SBP). (b) Ascitic fluid (AF) suPAR
concentrations were highest in the presence of culture-positive (n = 13) and culture-negative (n = 17) SBP, but did not differ
between sterile ascites without bacterial DNA fragments (bactDNA; n = 85), sterile ascites with bactDNA (n = 39), and
monomicrobial non-neutrocytic bacterascites (n = 8). The mean and SEM are indicated and the P value from the Kruskal–
Wallis test is shown [*P < 0.05 and **P < 0.01 (Dunn’s post hoc test)]. (c) SuPAR concentrations in AF correlated with local
concentrations of lipopolysaccharide-bind protein (LBP) in patients with SBP. Spearman’s rank correlation coefficient is
indicated. (d) AF suPAR levels in patients who died within 28 days after SBP were higher than patients who survived or
underwent liver transplantation (LTX) or died without presenting with SBP. The median and P values from the Mann–
Whitney U-test are indicated. (e) Kaplan–Meier analysis of survival indicates the highest mortality in patients with SBP and
AF suPAR levels 13.9 ng mL 1 (black dashed line, n = 14) compared to patients with SBP and lower AF suPAR levels
(black solid line, n = 16) and to patients without SBP (grey solid line, n = 132). P value from the log-rank test is indicated. (f)
Patients with SBP and AF suPAR levels 13.9 ng mL 1 presented numerically more often with hypotension (systolic blood
pressure < 90 mmHg), decreased renal function (increase in serum creatinine 0.3 mg dL 1 compared with hospitalization
or recent baseline values) and acute kidney injury according to the AKIN criteria (increase in serum creatinine
0.3 mg dL 1 within 48 h after inclusion).
94
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
H. W. Zimmermann et al.
CI = 0.59–0.81; P = 0.001) and culture-positive
SBP at an AUROC of 0.83 (95% CI = 0.71–0.94;
P < 0.001).
In 39 patients with sterile, non-neutrocytic ascites,
fragments of bacterial DNA were detected, which
indicated bacterial translocation (Table S1). The
median levels of AF suPAR did not differ between
bacterial deoxyribonucleic acid (bactDNA)-positive
and bacterial deoxyribonucleic acid (bactDNA)negative patients and were comparable to patients
with non-neutrocytic monomicrobial bacterascites
(Fig. 2b). In contrast to the circulating suPAR
concentration, the AF concentrations were largely
independent of the liver function scores (Table 2).
The AF suPAR levels were higher in 28-day nonsurvivors after SBP (Fig. 2d), with 13.9 ng mL 1 as
the best cut-off based on ROC analysis [area under
curve (AUC) = 0.65; sensitivity = 65%; specificity = 77%]. The cumulative 28-day survival rates
were 63% in patients with SBP and AF suPAR
concentrations <13.9 ng mL 1 and 21% in patients
with AF suPAR concentrations >13.9 ng mL 1
[P = 0.07 (log-rank test); Fig. 2e]. Although higher
AF suPAR concentrations identified patients with
an increase in serum creatinine of >0.3 mg dL 1
from baseline (P = 0.03), elevated AF suPAR concentrations failed to predict subsequent acute kidney injury according to the AKIN criteria (P = 0.46;
Fig. 2e). Furthermore, six of 14 patients (43%) with
an AF suPAR 13.9 ng mL 1 presented with
hypotension (systolic blood pressure <90 mmHg),
indicating a more severe course of SBP (Fig. 2e).
Monocytes and neutrophils release suPAR in response to ligation of
TLRs
Increased levels of circulating suPAR are thought
to correlate with cellular immune activation [21].
Flow cytometry of peripheral blood and AF demonstrated that amongst leucocyte subsets, only
monocytes, neutrophils, and CD14+ peritoneal
macrophages had detectable levels of membranebound uPAR (CD87) as a relevant potential source
of suPAR (Fig. 3a). Based on cultures of circulating
leucocyte populations isolated from healthy volunteers, monocytes released more soluble uPAR
(719 62 pg per 106 cells) than neutrophils
(224 108 pg per 106 cells) within 24 h (P =
0.008).
TNF-a and LPS, which are important mediators of
immune cell activation and inflammation in cir-
suPAR in advanced cirrhosis
rhosis, have been shown to up-regulate uPAR
expression on neutrophils and monocytes in vitro
and in vivo [31–33], but little is known about the
contribution of these cells to circulating suPAR
levels. Stimulation of monocytes with LPS at a
concentration of 10 ng mL 1 resulted in a 2–4-fold
increase in soluble uPAR in the supernatant,
whereas TNF-a did not have a comparable effect
in doses up to 100 ng mL 1 (Fig. 3b,c). Compared
with monocytes, cultured neutrophils released less
suPAR in response to LPS and TNF-a (Fig. 3b).
Having determined that TLR4 activation by LPS is a
potent stimulus for suPAR release from monocytes,
we next determined whether or not engagement of other
TLRs had similar effects. Stimulation of monocytes
and neutrophils with zymosan, a TLR2 agonist, and
of monocytes with poly I:C, a TLR3 agonist, also
resulted in increased suPAR release, but did not
exceed the effect observed with LPS (Fig. 3c).
Early shedding of membrane-bound uPAR from monocytes by LPS is
followed by uPAR recovery on the surface
To elucidate the possible mechanisms underlying
suPAR release, we stimulated monocytes with LPS
(10 ng mL 1) and determined the expression of
uPAR at different time-points. After 2 h of stimulation, the expression of uPAR was markedly
reduced, suggesting initial cleavage. At the 4 and
8 h time-points, however, we observed that uPAR
reappeared on the cell surface, and after 24 h
uPAR exceeded basal expression (Fig. 3d). Monocytes treated with LPS and Brefeldin A, which
inhibits translocation of secretory proteins from
the endoplasmatic reticulum to the Golgi [34],
failed to re-express uPAR on the cell surface and
released less soluble uPAR into the cell culture
medium, indicating de novo synthesis of uPAR
after LPS treatment (Fig. 3d). Culture of monocytes
in serum-free medium did not abrogate suPAR
release upon stimulation with LPS, demonstrating
that proteolytic serum enzymes are not essential
for suPAR shedding.
Discussion
We have reported that circulating suPAR levels in
patients with advanced cirrhosis increase with
clinical decompensation, correlate with worsening
liver function, and indicate poor short-term survival independent of infections and sepsis, whereas
AF suPAR concentrations are suggestive of SBP
and are largely independent of blood suPAR levels
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
95
H. W. Zimmermann et al.
suPAR in advanced cirrhosis
peritoneal infiltrate is responsible for the local level.
In agreement with our results, increased local levels
of suPAR in cerebrospinal fluid during bacterial
meningitis [35] and in bronchoalveolar lavage specimens from patients with inhalation trauma [36] are
associated with the extent of local inflammation.
Mesothelial cells have been shown to release suPAR
and liver function. These findings suggest a compartmental regulation of suPAR in which blood
levels reflect liver disease and AF levels indicate the
presence and extent of local infection.
The AF levels of suPAR were only elevated in the
presence of active infection, implying that the
uPAR
IgG1
Ascitic fluid
uPAR
Peripheral blood
800
600
400
10
10 1
10 2
3
CD8
7 (PE 10
)
80
96.1%
IgG1
uPAR
60
40
20
200
0
100
CD14 (PerCP-Cy5)
Neutrophils
Monocytes
Lymphocytes
96.1
1000
SSC-A
IgG1
uPAR
IgG1
% of max
(a)
57.2%
0
0
0
200
400
600
800
1000
Isotype (APC-Cy7)
Phycoerythrin
FSC-A
10
4
(b)
(c)
P
P
.
–1
ng mL
100
(d)
100
0h
80
2h
–1
ng mL
particles
–1
cell
100
80
–1
ng
–1
mL
ng mL
4h
–1
ng mL
particles
–1
cell
Monocyte uPAR expression
Medium only
80
40
20
0
40
24 h
uPAR (PE)
IgG 1
uPAR (PE)
24 h
Soluble uPAR release
10 ng mL–1 LPS
60
10 ng mL–1 LPS plus
Brefeldin A
40
0
uPAR (PE)
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
Isotype control
Medium only
20
20
40
0
uPAR (PE)
80
60
10 ng mL–1 LPS plus
Brefeldin A
60
20
100
8h
% of max
% of max
40
0
uPAR (PE)
80
96
60
20
100
0
% of max
% of max
% of max
10 ng mL–1 LPS
60
H. W. Zimmermann et al.
in response to LPS and inflammation [37]. It is
possible that mesothelial cells contributed to
suPAR levels in our patient cohort; however, AF
suPAR was not elevated in patients with bacterascites or the presence of bactDNA and correlated
with AF LBP levels only in the presence of infiltrating inflammatory cells, suggesting that activated
leucocytes are the main source of suPAR. Moreover,
our prospective data suggest that AF suPAR may be
clinically useful in predicting complicated cases of
SBP, which warrant escalation of treatment.
In contrast to AF suPAR, circulating levels of
suPAR were elevated in patients with decompensated cirrhosis without evidence of bacterial infection or sepsis. These patients presented with
median suPAR levels of 12.9 ng mL 1, which
exceeded the level that predicts SIRS (2.8 ng mL 1)
[38] or severe sepsis (6.6 ng mL 1) [17] in noncirrhotic patients, showing that high circulating
suPAR levels are associated with liver disease per
se, and not merely a reflection of sepsis in patients
with advanced cirrhosis. Circulating suPAR correlates with clinical decompensation, decreased liver
function, and markers associated with liver-related
mortality in cirrhosis, such as creatinine [6],
sodium [39], CRP [10], IL-10 [40, 41], and midregional proadrenomedullin [42]. Despite a good
correlation with suPAR and the MELD score, only
19% (rs2) of the variance of suPAR was explained by
the MELD score alone, indicating valuable information beyond liver function. In fact, serum suPAR
as a single continuous parameter predicted 28-day
mortality slightly better than the composite MELD
score. Patients with serum suPAR levels
>14.4 ng mL 1 were three times as likely to die
within 28 days and twice as likely to die within
suPAR in advanced cirrhosis
90 days compared with patients with lower serum
concentrations of suPAR, even after adjusting for
the MELD score, overt bacterial infection, and
co-morbidities.
Although activated neutrophils contribute to
suPAR during bacterial infection and inflammation
[11, 43, 44] and likely release local suPAR in SBP,
our data suggest that monocytes are the major
source of the circulating suPAR in patients with
advanced liver disease. When activated via TLRs,
monocytes secreted 5–10-fold more suPAR than
neutrophils. Surface uPAR is rapidly cleaved from
monocytes in the presence of LPS, followed by reexpression and continuing release [33]. Blocking
cytoplasmic protein transport resulted in abrogated uPAR expression and release, indicating that
continued cleavage and up-regulation of uPAR are
necessary for ongoing release from monocytes.
Thus, we believe that activated monocytes and
liver-resident macrophages are the major source of
circulating suPAR in patients with cirrhosis, even
in the absence of overt infection and SIRS. We and
others [27, 45, 46] have previously shown activation of circulating and hepatic monocytes with
progressive fibrosis, cirrhosis, and portal hypertension, and our current data have demonstrated a
correlation between serum suPAR with serum IL10, which is mainly derived from monocyte progeny
[47]. Furthermore, endotoxaemia and bacterial
translocation are characteristic of decompensated
cirrhosis, thus providing a source of TLR agonists
in patients with advanced cirrhosis independent of
overt infection [28, 30, 48]. The presence of large
numbers of activated monocytes and bacterial
translocation in advanced cirrhosis probably
Fig. 3 uPAR expression and suPAR release from monocytes and neutrophils. (a) Representative flow cytometry analysis of
peripheral blood and ascitic fluid demonstrating uPAR (CD87) expression on circulating monocytes (left panel, dark grey)
and neutrophils (left panel, black), as well as resident peritoneal macrophages (right panel, light grey), in contrast to
lymphocytes (left panel, light grey). The isotype-matched controls on the respective populations are indicated. (b) Release of
suPAR from freshly isolated primary human monocytes or neutrophils into cell culture media within 24 h after in vitro
stimulation with combinations of lipopolysaccharide (LPS) at 10 ng mL 1 and tumour necrosis factor-alpha (TNF-a) at
10 ng mL 1. The mean and SEM of at least three independent experiments are shown. P values derived from repeated
measures ANOVA [*P < 0.05 and ***P < 0.001 (Dunnet’s post hoc test)]. (c) Dose-dependent suPAR release from freshly
isolated primary human monocytes or neutrophils into cell culture media within 24 h after in vitro stimulation with TNF-a
and various Toll-like receptor (TLR) agonists. TNF-a was used in concentrations up to 100 ng mL 1, LPS up to 100 ng mL 1,
zymosan up to 10 particles per immune cell and polyinosinic/polycytidylic acid (poly I:C) at 10 lg mL 1. (d) uPAR
expression on freshly isolated monocytes after different times of incubation with medium alone (light grey area), 10 ng mL 1
of LPS (black solid line), or 10 ng mL 1 of LPS with 0.4 lL Brefeldin A (black dashed line), indicating early cleavage of uPAR
from the surface, beginning recovery after 4 h and overexpression after 8 h. The isotype-matched control is indicated at
0 h (grey dashed line). One representative plot of three independent experiments is shown. Bar diagrams show the
corresponding suPAR release from monocytes or neutrophils into culture medium over 24 h, indicating that up-regulation of
uPAR is necessary for significant suPAR release after initial cleavage.
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
97
H. W. Zimmermann et al.
explains why circulating suPAR levels are far
higher than the circulating suPAR levels in other
patients with suspected infection (6.4 ng mL 1)
[17], bacteraemia (9–11 ng mL 1) [14–16], or
sepsis (8–12 ng mL 1) [11, 12].
Beyond the role of suPAR as an indicator of liverrelated short-term mortality and immune activation, what is the role of suPAR in liver disease?
There is evidence that elevated suPAR may be
harmful because depletion of suPAR ameliorates
glomerular damage and restores kidney function in
rodent models of focal segmental glomerulosclerosis
[49], and perhaps surprisingly in murine E. coli
peritonitis uPAR-deficient animals have a lower
peritoneal and circulating bacterial load [50]. Thus,
suPAR depletion may be a potential therapeutic
approach in patients with advanced cirrhosis.
Conflict of interest statement
No conflicts of interest to declare.
Acknowledgements
The authors would like to thank Aline Roggenkamp
for excellent technical assistance.
Contributions
HWZ, FT, and TB designed the study, performed
the in vitro experiments, performed statistical
analysis, analysed and interpreted the results,
conducted the literature search and wrote the
manuscript. HWZ, PAR, MB, FT and TB recruited
patients, collected specimens, performed measurements and analysed clinical data. AK, DHA, CT and
AS participated in the design of the study, interpreted the results and revised the manuscript. All
authors read and approved the final version of the
manuscript.
Funding
This work was supported, in part, by the Federal
Ministry of Education and Research (BMBF) Germany (FKZ: 01 E0 1002). The German Research
Foundation provided research funding to TB (BR
4182/1-1), FT (DFG Ta434/2-1 and SFB/TRR 57),
and CT (SFB/TRR 57). HWZ and FT received
funding from the START-Program and the Interdisciplinary Centre for Clinical Research (IZKF)
within the Faculty of Medicine at the RWTH Aachen
98
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
suPAR in advanced cirrhosis
University. The suPARnosticTM ELISA kits were gifts
from ViroGates (Birkeroed, Denmark).
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Correspondence: Tony Bruns, NIHR Biomedical Research Unit
and Centre for Liver Research, Institute of Biomedical Research,
University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
(fax: +44 121 415 8701; e-mail: t.bruns@bham.ac.uk).
100
ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 86–100
suPAR in advanced cirrhosis
Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Data S1. Supplemental methods.
Table S1. Isolated microorganisms and identified
microbial DNA from ascitic fluid.