C-reactive Protein

CRP was first described in the early 1930s,

[18]
and CRP levels are
widely used as a relatively nonspecific marker of inflammation. Many
studies have now been published that demonstrated increased CRP
levels in patients with sepsis.
[1921]
As an acute phase protein, however,
CRP levels are increased to some extent in most conditions associated
with inflammation, including rheumatoid arthritis, Crohn's disease,
acute myocardial infarction and pancreatitis, to list but a few.
[22]
As
such, CRP is a very nonspecific marker of sepsis. Nevertheless, it is a
cheap and widely available test and when levels are raised in a patient
with signs suggestive of sepsis, it provides useful supporting evidence.
In 112 ICU patients, Pvoa et al.reported that a serum CRP
concentration of >8.7 mg/dl had a sensitivity of 93.4% and a specificity
of 86.1% for infection;
[19]
the combination of CRP >8.7 mg/dl and
temperature >38.2C increased the specificity for infection diagnosis to
100%.
Because baseline CRP levels are often raised as a result of comorbid
chronic inflammatory conditions, changes in concentrations over time
are more useful than single values.
[2325]
Pvoa et al.
[23]
showed that
CRP concentrations increased over time in infected patients, but
remained unchanged in non-infected patients. A maximum daily CRP
variation of greater than 4.1 mg/dl predicted nosocomial infection with a
sensitivity of 92.1% and a specificity of 71.4%; moreover, when
combined with a CRP concentration greater than 8.7 mg/dl, the
sensitivity and specificity increased to 92.1 and 82.1%, respectively.
Similarly, as a prognostic marker, a decrease in CRP level after 48 h
was associated with a mortality rate of 15.4%, while an increased CRP
level was associated with a mortality rate of 60.9% in patients with CRP
concentrations >10 mg/dl on ICU admission (p < 0.05).
[24]

Because CRP levels decrease as sepsis resolves, it has been
suggested that levels could be used to guide antimicrobial therapy. In
50 adult ICU patients with sepsis, Schmit & Vincent
[26]
reported that an
increase in CRP of at least 2.2 mg/dl in the first 48 h was associated
with ineffective initial antibiotic therapy with a sensitivity of 77% and a
specificity of 67%. Similarly, in patients with ventilator-acquired
pneumonia, serum CRP levels were significantly lower in patients with
appropriate antibiotic treatment than in those with inappropriate
empirical treatment at 96 h (10.3 10 mg/dl vs 19.2 14 mg/dl; p <
0.05),
[27]
and in patients with community-acquired pneumonia, a less
than 60% decrease in CRP levels by 3 days after admission (odds ratio
[OR]: 6.98; 95% CI: 1.5631.33) was associated with an increased risk
of having received inappropriate empiric antibiotic
treatment.
[28]
However, use of CRP levels to guide therapy has not been
tested prospectively in adult patients with sepsis.
Procalcitonin
PCT was described more recently than CRP and is not routinely
measured in all hospital laboratories. PCT levels have been shown to
be raised in patients with sepsis
[29,30]
and may be particularly useful in
distinguishing bacterial from other forms of infection.
[31]
In patients with
community-acquired pneumonia, PCT values >0.25 g/l had a
sensitivity of 96% and specificity of 40% for predicting
bacteremia.
[32]
Similarly, in patients with febrile urinary tract infection
presenting to the emergency department, PCT values >0.25 g/l had a
sensitivity of 95% and specificity of 50% for a diagnosis of
bacteremia.
[33]
In patients admitted to the emergency department with
symptoms of systemic infection, a PCT threshold of 0.15 ng/ml had a
sensitivity of 75%, specificity of 79% and negative predictive value of
98% for a diagnosis of bloodstream infection.
[34]
Several studies have
suggested that PCT is a more reliable marker of sepsis than CRP.
[3538]

Higher PCT levels have been associated with increased mortality
rates.
[39,40]
In ICU patients with sepsis, Giamarellos-Bourboulis et
al.
[39]
reported mortality rates of 25.6% in those with PCT less than or
equal to 0.85 ng/ml, but 45.3% in those with PCT greater than 0.85
ng/ml (OR for death: 2.404; 95% CI: 1.3854.171; p = 0.002). As with
CRP, PCT levels are raised in other inflammatory conditions, including
pancreatitis, acute myocardial infarction and postcardiac surgery.
Trends in concentrations over time are again of more value than single
measurements. Karlsson et al.
[41]
reported that although PCT
concentrations did not differ between hospital survivors and
nonsurvivors, mortality rates were lower in patients whose PCT
concentration decreased by more than 50% in 72 h than in those in
whom levels decreased by less than 50% (12.2 vs 29.8%; p = 0.007).
Nevertheless, the precise role of PCT remains unclear with meta-
analyses reporting conflicting findings. Indeed, one review concluded
that 'Procalcitonin should be included in diagnostic guidelines for sepsis
and in clinical practice in intensive care units',
[36]
whereas another
published a year later noted that 'The findings do not lend support to
the widespread use of the procalcitonin test in critical care
settings',
[42]
although differences in the search criteria and studies
included in these meta-analyses may explain these different
conclusions. The use of PCT levels to guide antibiotic therapy has been
tested in several clinical trials in different groups of infected patients.
However, despite reported beneficial effects of this approach on
antibiotic utilization in many of these studies,
[4347]
with no adverse
effects on patient outcomes, not all studies support these
findings.
[48]
Several meta-analyses of studies using this approach have
also been conducted. Schuetz et al.
[49]
recently reported, in a meta-
analysis of 14 trials in patients with acute respiratory infection, that use
of PCT to guide initiation and duration of antibiotic treatment was
effective in reducing antibiotic exposure in these patients without
increasing rates of treatment failure or mortality. Another meta-analysis
suggested that although PCT-guided antibiotic therapy was associated
with a reduction in antibiotic usage and reduced costs, an associated
7% increase in hospital mortality could not be excluded.
[50]

Cytokine Levels
Various cytokines, key mediators of the sepsis response, have been
assessed for their role as biomarkers of sepsis. Serum levels of IL-6,
TNF and IL-8, to mention just a few, have been shown to be increased
in patients with sepsis.
[29,51,52]
Raised serum levels have also been
shown to be associated with development of organ function and
mortality.
[51,53]
These cytokines are all involved in the inflammatory
response to infection, but levels are also increased in patients with
other inflammatory processes, such as arthritis and myocardial
infarction. Several recent studies have used multiplex technology to
measure multiple cytokine levels simultaneously. In a multicenter cohort
study of 60 patients with severe sepsis, Bozza et al.
[54]
evaluated the
prognostic value of 17 cytokines (IL-1, -2, -4, -5, -6, -7, -8, -10, -12, -
13, -17, IFN-, granulocyte colony-stimulating factor [G-CSF],
granulocytemacrophage colony-stimulating factor, MCP-1, MIP-1 and
TNF-). Levels of cytokines were correlated with disease severity and
development of organ dysfunction. IL-1, -4, -6, -8, MCP-1 and G-CSF
had good accuracy for predicting early mortality, whereas IL-8 and
MCP-1 had the best predictive value for 28-day mortality. In multivariate
analysis, only MCP-1 was independently associated with prognosis
(OR for hospital mortality: 1.41; 95% CI: 1.021.93; p = 0.036). In 30
patients with sepsis, Mera et al.
[55]
also measured 17 cytokines and
reported that the initial levels of IL-8 were the most predictive for fatal
outcome. Levels of IL-1, -6, -8, -12, IFN-, G-CSF and TNF-
remained high in nonsurvivors. In a study of 126 emergency
department patients with SIRS or sepsis, in which levels of 22 cytokines
were analyzed, only soluble IL-2 receptor (sIL-2R) levels were
independently associated with a diagnosis of severe sepsis. IL-1, sIL-
2R and IL-8 levels were independently associated with a diagnosis of
septic shock.
[56]
However, using principal component analyses and
hierarchical clustering analyses, the authors demonstrated that there
were no typical cytokine profiles associated with a diagnosis of severe
sepsis or septic shock.
Soluble Triggering Receptor Expressed on Myeloid
Cells-1
Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is
a member of the immunoglobulin superfamily, the expression of which
is upregulated in the presence of bacteria or fungi.
[57,58]
Serum levels of
sTREM-1 are increased in patients with sepsis,
[5962]
and several studies
have suggested that they are more sensitive and specific for infection
than other markers, including CRP and PCT.
[59,60]
However, in
emergency department patients, the specificity and sensitivity for
diagnosis of sepsis were only 59 and 60%, respectively.
[63]
In addition, a
decrease in sTREM-1 levels over time was associated with a favorable
outcome,
[58]
suggesting a potential place in following response to
therapy. Levels of sTREM-1 have also been studied in other body
fluids. Raised sTREM-1 levels have been reported in the
bronchoalveolar lavage fluid of patients with pneumonia,
[64,65]
and
urinary levels have been associated with early diagnosis of sepsis,
disease severity and prognosis.
[66]
In logistic regression analysis,
urinary sTREM-1 levels were a risk factor for development of acute
kidney injury in patients with sepsis (OR: 1.02; Wald coefficient: 5.246;
p = 0.022).
[66]
In a recent study in emergency department patients with
sepsis who underwent early goal-directed resuscitation, sTREM-1
concentrations were significantly higher at admission and pre-early
goal-directed resuscitation in nonsurvivors than in survivors (514.1
pg/ml [interquartile range: 412.71749.5 pg/ml] vs 182.4 pg/ml
[interquartile range: 54.3327.0 pg/ml]; p = 0.001); PCT and CRP levels
were the same in the two groups.
[67]
In multivariate regression analysis,
sTREM-1 values at admission were independently associated with poor
prognosis (OR for 28-day mortality: 2.78; 95% CI: 1.1166.945; p =
0.028).
CD64
CD64 is a membrane glycoprotein, which binds the Fc part of IgG. The
expression of CD64 on the neutrophil membrane is negligible in health,
but upregulated in response to proinflammatory cytokines as, for
example, in sepsis and other inflammatory conditions, such as arthritis.
Neutrophil CD64 expression has been reported to have variable
sensitivity and specificity for sepsis diagnosis and prognosis.
[63,6871]
In a
study of 132 emergency department patients with fever, 87% of whom
had bacterial infection, the CD4 index was higher in patients with
infection than in those without (3.7 3.2 vs 2.5 2.3; p = 0.03); the
area under the receiver operating characteristic curve (AUROC) for
detection of bacterial infection was 0.66 (95% CI: 0.520.8) and for
prediction of survival it was 0.71 (95% CI: 0.570.85).
[69]
In 163 infants
with suspected sepsis, the sensitivity and specificity of the CD4 index
using a cutoff of 2.30 were 70 and 62%, respectively; when combined
with an absolute neutrophil count of <7500 or >14,500 cells/mm
3
, the
sensitivity increased to 95% with a negative predictive value of
93%.
[70]
In 293 ICU patients with at least two SIRS criteria, using a
cutoff of 2.2, the CD64 index predicted bacterial infection with a
sensitivity and specificity of 63 and 89%, respectively, and positive and
negative predictive values of 85 and 70%, respectively.
[71]
In 631
emergency department patients with suspected sepsis, the sensitivity of
CD4 for diagnosing sepsis was 66% and the specificity 65% with
positive and negative predictive values of 79 and 49%, respectively.
Clot Waveform Analysis
In patients with sepsis, the optical transmission waveform obtained
during measurement of the activated partial thromboplastin time is
altered, giving a biphasic pattern. The presence of this abnormal
waveform has been used to support a diagnosis of sepsis.
[72]
In 331 ICU
patients, 59.3% of patients with a biphasic waveform on admission and
45.3% with a biphasic waveform during the total ICU stay developed
sepsis. The sensitivity of the waveform analysis for detection of sepsis
was poor, varying between 22 and 55% at admission and between 48
and 74% during the entire ICU stay, depending on the threshold value
of the slope used. The specificity for sepsis, however, was good,
varying between 92 and 98% at admission and between 81 and 94%
during the total ICU stay.
[73]
In 187 patients with SIRS, presence of a
biphasic waveform (90% sensitivity, 92% negative predictive value) was
better than PCT (71% sensitivity, 67% negative predictive value) or
CRP (88% sensitivity, 89% negative predictive value) for diagnosis of
severe sepsis and septic shock.
[74]
The biphasic waveform on day 3
also had better specificity (91%) and negative predictive value (98%)
for prediction of 28-day sepsis-related mortality (CRP specificity 57%,
negative predictive value 95%; PCT specificity 83%, negative predictive
value 95%).
[74]
Combination of an abnormal waveform plus a PCT level
>1 ng/ml on admission to the ICU was associated with a sensitivity of
79% and specificity of 96%, with a negative predictive value of 96%.
[75]

Growth Arrest-specific Protein 6
Gas6 is an intracellular vitamin-K dependent protein released by
leukocytes and endothelial cells in response to injury. Gas6 has roles in
cell survival (antiapoptotic), proliferation, migration and adhesion.
[76,77]
In
a matched casecontrol study, plasma Gas6 levels were higher in
patients with severe sepsis (median: 110 [interquartile range: 75139]
ng/ml) than in those with organ failure unrelated to infection (median:
85 [56101] ng/ml; p = 0.026) and in healthy control subjects (median:
54 [4968] ng/ml; p = 0.0001).
[77]
In the patients with severe sepsis,
Gas6 levels correlated disease severity as assessed using the
APACHE II score (Spearman correlation coefficient: 0.45; p = 0.012)
and the Organ Dysfunction and Infection (ODIN) score (nonparametric
test for trend, p = 0.030).
[77]
In 232 patients with fever and suspected
infection, Gas6 levels were significantly higher in patients with sepsis
than in controls (healthy blood donors; p < 0.001); levels were also
significantly higher in patients with severe sepsis than in those with
sepsis (p < 0.001).
[78]
In 45 ICU patients with septic shock, Gas6 levels
correlated with the Sequential Organ Failure Assessment (SOFA) score
(Spearman rank-order coefficient: 0.37; p = 0.01) and with hepatic
function as assessed by aspartate transaminase concentrations
(Spearman rank-order coefficient: 0.42; p = 0.006) and prothrombin
time (Spearman rank-order coefficient: 0.45; p = 0.02). Gas6
concentrations were also higher in patients who required renal support
than in those who did not (median: 76.5 [52164] pg/ml vs 10.5 [1.5
80.5] pg/ml; p = 0.04). Gas6 levels were also strongly correlated with
plasma PCT concentrations (Spearman rank-order coefficient: 0.44; p =
0.005). There were no differences in Gas6 levels among survivors and
nonsurvivors.
[76]

Angiopoietin
Angiopoietin (Ang)-1 and -2 are endothelial-derived vascular growth
factors that have contrasting roles in endothelial activation during
sepsis: Ang-1 stabilizes the endothelium, whereas Ang-2 promotes loss
of barrier integrity and vascular leak.
[79]
Increased levels of Ang-2 have
been reported in patients with severe sepsis compared with those with
no evidence of inflammatory response (p < 0.05).
[80]
In patients with
sepsis, Ang-2 levels correlated with disease severity as measured by
the APACHE II (r = 0.395; p = 0.007) or SOFA (r = 0.402; p = 0.006)
scores and were higher in nonsurvivors than in survivors (24.9 ng/ml
[21.538.0] ng/ml vs 13.5 [8.121.6] ng/ml; p = 0.02).
[81]
In a study of 70
patients with severe sepsis, survivors had higher peak Ang-1 levels
(median: 13 vs 10 ng/ml; p = 0.019) and lower nadir Ang-2 levels
(median: 2.8 vs 6.2 ng/ml; p = 0.013) than did nonsurvivors.
[79]
Lower
Ang-1 levels (<5.5 ng/ml) on admission were independently associated
with an increased risk of death at 28-days compared with higher Ang-1
levels (OR: 0.282; 95% CI: 0.0860.93; p = 0.03).
[79]
Raised levels of
Ang-2 have also been reported in patients with trauma and after
cardiopulmonary bypass surgery.
Soluble Urokinase-type Plasminogen Receptor
Urokinase-type plasminogen receptor (uPAR) is expressed on various
cell types, including neutrophils, lymphocytes, monocytes,
macrophages and vascular endothelial cells. uPAR is involved in
multiple immunological functions, including migration, adhesion,
angiogenesis, fibrinolysis and cell proliferation.
[82,83]
During inflammatory
disease processes, such as sepsis, uPAR is cleaved from the cell
surface by proteases, releasing a soluble form of the receptor, soluble
uPAR (suPAR). Elevated levels of suPAR have been reported in
patients with sepsis compared with patients without (on admission,
median: 11.05 [1.8720] ng/ml vs 7.62 [020] ng/ml; p <
0.001).
[82]
However, in this prospective study of 273 critically ill patients,
197 of whom had sepsis, the AUROC for prediction of sepsis was only
0.62, compared with 0.86 for CRP and 0.78 for PCT. In 151 patients
with SIRS and suspected community-acquired sepsis, 96 of whom had
bacterial infections, the AUROC for diagnosis of bacterial sepsis was
0.50 for suPAR, 0.72 for PCT and 0.81 for CRP, again suggesting that
suPAR had lower diagnostic capability than did these other
biomarkers.
[84]
In an ongoing study in critically ill patients, a cutoff suPAR
value of 5.5 ng/ml had a sensitivity of 75% and a specificity of 72% for
diagnosing sepsis (AUROC: 0.75; 95% CI: 0.660.83).
[85]
Several
studies have suggested that suPAR may be of more value as a
prognostic marker than for diagnosis.
[82,83,86,87]
In 543 acutely ill medical
patients, baseline suPAR levels were significantly lower in 30-day (4.97
ng/ml vs 7.31 ng/ml; p = 0.0002) and 90-day (4.87 ng/ml vs 7.29 ng/ml;
p < 0.0001) survivors than in nonsurvivors.
[87]
Increased suPAR levels
remained significantly associated with 30-day mortality (hazard ratio
[HR]: 1.10; 95% CI: 1.011.20) and 90-day (HR: 1.11; 95% CI: 1.04
1.19) after adjusting for age, sex, CRP levels and the Charlson Score.
In a recent meta-analysis of six studies that had assessed the
prognostic role of suPAR, suPAR had better prognostic ability and was
superior in predicting mortality than other commonly used markers,
including CRP, PCT and sTREM-1, although it performed less well than
the SAPS II score.
[86]
In another recent study, Giamarellos-Bourboulis et
al. developed and validated a mortality risk prediction rule with four
levels of risk, using combinations of APACHE II values (<17 or >17)
and serum suPAR concentrations (<12 ng/ml or >12 ng/ml). The score
had a negative predictive value of 94.5% in patients with sepsis.
[88]

HLA
The above mentioned biomarkers have largely been indicative of the
proinflammatory phase of sepsis, but sepsis is also associated with an
anti-inflammatory state, which, if prolonged, can lead to
immunodepression or so-called immune 'paralysis'. Monocyte HLA-DR
is part of the major histocompatibility class II antigen complex. The
main role of HLA-DR in infection is in antigen presentation to T-helper
cells resulting in release of proinflammatory cytokines, but in patients
with sepsis, this pathway can be impaired leading to
immunodepression.
[89]
Several studies have suggested that HLA-DR
expression may be indicative of prognosis. In an early study in surgical
patients with sepsis, Volk et al. reported a survival rate of 81% in
patients with normal monocyte HLA-DR expression, but only 19% in
patients with a persistently (at least 5 days) reduced HLA-DR
expression of <30%.
[90]
More recently, in patients with septic shock,
Monneret et al. also demonstrated significant differences in HLA-DR
expression in survivors and nonsurvivors; the percentage of monocytes
expressing HLA-DR was reduced in all patients during the first 48 h of
septic shock, but on days 34 the percentage increased in survivors but
not in nonsurvivors (43 vs 18%; p < 0.001).
[91]
In multivariate logistic
regression analysis, low monocyte HLA-DR expression (<30%) at days
34 was significantly associated with mortality after adjustment for
other confounders with an adjusted OR of 6.48 (95% CI: 1.6225.93).
In a recent retrospective study, Trimmel et al. were, however, unable to
identify a specific cutoff value for HLA-DR expression that was
correlated with mortality, raising questions about the value of absolute
values for risk prediction.
[92]
Supporting this, in patients with severe
sepsis, Wu et al. reported that there were no differences between
survivors and nonsurvivors using a 30% cutoff for HLA-DR expression;
however, a change in monocyte HLA-DR expression of 4.8% over the
first 3 days of ICU admission did allow discrimination between survivors
and nonsurvivors with a sensitivity of 89.0% and a specificity of
93.7%.
[93]