Timing of adequate antibiotic therapy is a greater determinant of outcome than are TNF and IL-10 polymorphisms in patients with sepsis.

Abstract

Genetic variations may influence clinical outcomes in patients with sepsis. The present study was conducted to evaluate the impact on mortality of three polymorphisms after adjusting for confounding variables, and to assess the factors involved in progression of the inflammatory response in septic patients.
The inception cohort study included all Caucasian adults admitted to the hospital with sepsis. Sepsis severity, microbiological information and clinical variables were recorded. Three polymorphisms were identified in all patients by PCR: the tumour necrosis factor (TNF)-alpha 308 promoter polymorphism; the polymorphism in the first intron of the TNF-beta gene; and the IL-10-1082 promoter polymorphism. Patients included in the study were followed up for 90 days after hospital admission.
A group of 224 patients was enrolled in the present study. We did not find a significant association among any of the three polymorphisms and mortality or worsening inflammatory response. By multivariate logistic regression analysis, only two factors were independently associated with mortality, namely Acute Physiology and Chronic Health Evaluation (APACHE) II score and delayed initiation of adequate antibiotic therapy. In septic shock patients (n = 114), the delay in initiation of adequate antibiotic therapy was the only independent predictor of mortality. Risk factors for impairment in inflammatory response were APACHE II score, positive blood culture and delayed initiation of adequate antibiotic therapy.
This study emphasizes that prompt and adequate antibiotic therapy is the cornerstone of therapy in sepsis. The three polymorphisms evaluated in the present study appear not to influence the outcome of patients admitted to the hospital with sepsis.

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Vol 10 No 4Research
Timing of adequate antibiotic therapy is a greater determinant of
outcome than are TNF and IL-10 polymorphisms in patients with
sepsis
Jose Garnacho-Montero1, Teresa Aldabo-Pallas1, Carmen Garnacho-Montero2, Aurelio Cayuela3,
Rocio Jiménez1, Sonia Barroso1 and Carlos Ortiz-Leyba1
1Intensive Care Unit, Hospital Universitatrio Virgen del Rocio, Seviilla, Spain
2Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
3Supportive Research Unit, Hospital Universitario Virgen del Rocio, Sevialla, Spain
Corresponding author: Jose Garnacho-Montero, jgmrji@arrakis.es
Received: 21 Feb 2006 Revisions requested: 19 Apr 2006 Revisions received: 29 Jun 2006 Accepted: 18 Jul 2006 Published: 19 Jul 2006
Critical Care 2006, 10:R111 (doi:10.1186/cc4995)
This article is online at: http://ccforum.com/content/10/4/R111
© 2006 Garnacho-Montero et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Introduction Genetic variations may influence clinical outcomes
in patients with sepsis. The present study was conducted to
evaluate the impact on mortality of three polymorphisms after
adjusting for confounding variables, and to assess the factors
involved in progression of the inflammatory response in septic
patients.
Method The inception cohort study included all Caucasian
adults admitted to the hospital with sepsis. Sepsis severity,
microbiological information and clinical variables were recorded.
Three polymorphisms were identified in all patients by PCR: the
tumour necrosis factor (TNF)-α 308 promoter polymorphism;
the polymorphism in the first intron of the TNF-β gene; and the
IL-10-1082 promoter polymorphism. Patients included in the
study were followed up for 90 days after hospital admission.
Results A group of 224 patients was enrolled in the present
study. We did not find a significant association among any of the
three polymorphisms and mortality or worsening inflammatory
response. By multivariate logistic regression analysis, only two
factors were independently associated with mortality, namely
Acute Physiology and Chronic Health Evaluation (APACHE) II
score and delayed initiation of adequate antibiotic therapy. In
septic shock patients (n = 114), the delay in initiation of
adequate antibiotic therapy was the only independent predictor
of mortality. Risk factors for impairment in inflammatory
response were APACHE II score, positive blood culture and
delayed initiation of adequate antibiotic therapy.
Conclusion This study emphasizes that prompt and adequate
antibiotic therapy is the cornerstone of therapy in sepsis. The
three polymorphisms evaluated in the present study appear not
to influence the outcome of patients admitted to the hospital
with sepsis.
Introduction
Mortality from sepsis remains unacceptably high despite
recent advances in diagnostic procedures, antimicrobial treat-
ment, and supportive care [1,2]. Although antibiotic therapy is
the cornerstone of treatment of infections, the influence of
adequate antimicrobial therapy on prognosis in septic patients
was not clearly proven until recently. We and others demon-
strated that after controlling for confounding variables, ade-
quate empirical antibiotic treatment is associated with
reduced mortality in critically ill septic patients [3-5]. Interest-
ingly, the impact on outcome of delayed adequate antibiotic
therapy has not been studied in septic patients. The variables
that can influence outcome in septic patients are numerous
(such as, age, underlying disease, source of sepsis, presence
of bacteraemia and organ system dysfunction). In addition,
several interventions have been shown to decrease mortality in
sepsis and should be taken into account when analyzing the
impact on outcome of any single factor.
APACHE = Acute Physiology and Chronic Health Evaluation; bp = base pair; CI = confidence interval; ICU = intensive care unit; IL = interleukin; OR
= odds ratio; PCR = polymerase chain reaction; SOFA = Sequential Organ Failure Assessment; TNF = tumour necrosis factor.

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At the beginning of the third millennium, much interest and
great expectation were focused on advances in molecular biol-
ogy, and particularly on the completion of Human Genome
Project. Individual variants of genes encoding mediators that
are involved in the inflammatory response to an infectious
agent might account for differences in clinical evolution and
outcome of septic patients treated correctly and with appar-
ently similar approaches. Tumour necrosis factor (TNF) is con-
sidered the most important proinflammatory cytokine,
recruiting and activating immune cells, stimulating the release
of other proinflammatory mediators and regulating apoptosis.
In contrast, IL-10 is the paradigmatic anti-inflammatory
cytokine. It exerts its biological properties by inhibiting the
release of proinflammatory cytokines and preventing apopto-
sis. However, contradictory observations have been reported
on the impact on outcome of polymorphisms in these
cytokines that are directly responsible for the host response
[6,7].
An international panel of experts defined systemic inflamma-
tory response syndrome, sepsis, severe sepsis and septic
shock, which are viewed as a continuum of risk [8]. Factors
that influence the progression of inflammatory response in
patients admitted to the hospital because of sepsis have not
been elucidated. In other words, the causes why clinical situa-
tion deteriorates are not clearly known.
Hence, the primary aim of the present study, conducted in
adults admitted to the hospital with sepsis, was to evaluate the
impact on in-hospital mortality (and 90-day mortality) of three
polymorphisms (the TNF-α-308 promoter polymorphism, the
polymorphism in the first intron of the TNF-β gene, and the IL-
10-1082 promoter polymorphism), after controlling for various
confounding variables including delayed adequate antibiotic
therapy. We chose these mediators because an altered bal-
ance in their serum concentrations has been associated with
poor outcome in patients with community-acquired infection
[9], and we selected these specific polymorphisms because of
their functional significance [10-12]. Our secondary objec-
tives were to explore this same objective in patients with septic
shock and to assess the factors that are involved in progres-
sion of the inflammatory response.
Materials and methods
Hospital
This prospective study was carried out in the Hospital Virgen
del Rocío – a large university hospital with a 40-bed intensive
care unit (ICU) – from June 2002 to December 2004. Written
informed consent was obtained from patients or their relatives,
and the ethics committee of the hospital approved the study.
Patients
Eligible patients were Caucasian adults (age >18 years) who
arrived in at the emergency department meeting criteria for
sepsis. Infected patients who did not fulfill sepsis criteria were
not included. The criteria followed for ICU admission were
based on the patient clinical condition, being shock or respira-
tory insufficiency the main reasons for ICU admission. Patients
not admitted to the ICU were transferred to the general ward.
The exclusion criteria were neutropenia (white blood cell count
<500/µl), positive HIV serologic results and pregnancy.
Patients included in the protocol were followed up until death
or hospital discharge. Vital status of those patients discharged
from the hospital before 90 days was ascertained by search-
ing in the hospital database or telephone contact. The control
group comprised 101 healthy unrelated blood donors from the
hospital blood bank.
Study design
All patients diagnosed with sepsis received standard support-
ive treatment, including prompt fluid resuscitation, vasoactive
drugs and empirical antimicrobial therapy, which was chosen
by the attending physician [3]. Intravenous hydrocortisone
(200 mg/day for seven days) was used in patients in septic
shock who, despite fluid replacement, required vasopressor
agents [13], and continuous infusion of insulin was adminis-
tered to control blood glucose levels, in accordance with the
results of a large clinical trial [14]. All patients on mechanical
ventilation were managed following ARDSNet recommenda-
tions [15].
All patients had a series of blood cultures drawn at admission.
Sepsis was documented when a relevant micro-organism from
a suspected focus was isolated and/or bacteraemia was
present. Cultures of infection sources were obtained in all
patients as clinically indicated. Paired serum samples were
tested for evidence of antibody against respiratory viruses,
Legionella pneumophila, Chlamydia spp., Coxiella burnetii
and Mycoplasma pneumoniae. Tracheal aspirate or protected
brush specimen were obtained from all patients with commu-
nity-acquired pneumonia who required mechanical ventilation.
Variables
Sepsis, severe sepsis and septic shock were defined follow-
ing current definitions [8]. Severity of illness was evaluated
using the Acute Physiology and Chronic Health Evaluation
(APACHE) II score, recording the worst reading during the
first 24 hours in the hospital [16]. Chronic organ insufficien-
cies (liver, renal, pulmonary, cardiovascular and immunosup-
pression) were recorded as defined in the APACHE II scale,
and other comorbidities (alcoholism, smoking habit, diabetes
mellitus and noncured malignancy) as defined by Pittet and
coworkers [17]. Other variables recorded included bacterae-
mia, microbiologically documented infection and delay from
hospital admission (documented time when the patient arrived
at the emergency department) to administration of adequate
antibiotic therapy. Time elapsed from hospital admission to
onset of operation was noted in surgical patients. Empirical
therapy was considered adequate when at least one effective
drug was included in the antibiotic treatment regimen within

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the first 24 hours in the hospital, and the dose and pattern of
administration were in accordance with current standards.
Failure of organs was evaluated using the Sequential Organ
Failure Assessment (SOFA) scale on admission and during
the subsequent clinical course [18]. Worsening in the inflam-
matory response was monitored by two methods: we deter-
mined the proportion of patients admitted to the hospital with
sepsis who developed severe sepsis or septic shock and the
proportion of patients with severe sepsis at admission who
developed septic shock; and we calculated the delta-SOFA
(i.e. the worst SOFA score during hospitalization minus the
SOFA score during the first 24 hours) [19]. Nosocomial infec-
tions (pneumonia, catheter-related bloodstream infection and
primary bacteraemia) were diagnosed as previously defined
[20].
Genotyping
Genomic DNA from each patient was extracted from whole
blood using a DNA extraction Kit (Puregene DNA Isolation kit,
Minneapolis, MN, USA), in accordance with the manufac-
turer's instructions.
Polymorphism at TNF-α promoter position -308
DNA samples were amplified by PCR with forward primer
TNF-α-F (5'-AGG CAA TAG GTT TTG AGG GCC AT-3') and
reverse primer TNF-α-R (5'-ACA CTC CCC ATC CTC CCT
GCT-3'). The TNF-α primer includes a single base pair (bp)
mismatch, which introduces an NcoI restriction site after
amplification when the G allele is present at position -308. A
116 bp PCR product was obtained from a 50 µl reaction mix
containing 100 ng DNA, 1 µmol/l each primer, 0.2 mmol/l
dNTP, 1× buffer, 1.5 mmol/l MgCl2 and 1 U Taq polymerase
(Finnzymes, Espoo, Finland). The reaction was carried out with
the following cycles: 95°C for 2 minutes; 35 cycles of 95°C for
30 s, 60°C for 15 s and 74°C for 15 s; and 74°C for 10 min-
utes for final extension. The PCR product was incubated with
NcoI (New England Biolabs) and digested, and undigested
samples were visualized by electrophoresis in 4% Nu Sieve
agarose gel (Master Diagnostica, Madrid, Spain) and ethidium
bromide staining. A single band at 116 bp identified AA
homozygous individuals, two bands at 96 and 20 bp identified
GG homozygous individuals, and three bands at 116, 96 and
20 bp indicated a heterozygote at the TNF-α-308 locus.
Polymorphism in the first intron of the TNF-β gene (NcoI
polymorphism)
The region with the +250 polymorphism, which contains an
NcoI restriction site when the G allele is present, was amplified
using primers TNF-β-F (5'-CCG TGC TTC GTG CTT TGG
ACT A-3') and TNF-β-R (5'-AGA GGG GTG GAT GCT TGG
GTT C-3'). Each 50 µl reaction mix consisted of 100 ng DNA,
1 µmol/l each primer, 0.2 mmol/l dNTP, 1× buffer, 1.5 mmol/l
MgCl2 and 1 U Taq polymerase. Amplification was performed
with an initial denaturation of 95°C for 2 minutes; followed by
35 cycles of 94°C for 30 s, 69°C for 30 s and 74°C for 42 s;
and completing the reaction with a final extension step of 74°C
for 10 minutes. The 782 bp PCR product was digested with
the restriction enzyme NcoI (New England Biolabs), incubat-
ing at 37°C for two hours. The obtained fragments as well as
undigested samples were analyzed by electrophoresis in
1.5% agarose gel and visualized by ethidium bromide staining.
The presence of a single band of 782 bp identified individuals
who were AA homozygous, two bands at 586 and 196 bp indi-
cated those who were GG homozygous, and heterozygous
individuals were identified by three bands at 782, 586 and
196 bp.
Polymorphism at IL-10 promoter position -1082
IL-10-1082 polymorphism results from the substitution of gua-
nine with adenine, which abolishes a MnlI restriction site. The
region containing this polymorphism was amplified by PCR
using primers IL-10-F (5'-CTC GTC GCA ACC CAA CTG-3')
and IL-10-R (5'-ACT TTC ATC TTA CCT ATC CCT ACT TCC-
3'). The amplified region also includes the CA repeat micros-
atellites located at -1151 (IL-10-G polymorphism). A 50 µl
reaction mix contained 100 ng DNA, 0.5 µmol/l each primer,
0.2 mmol/l dNTP, 1× buffer, 1.5 mmol/l MgCl2 and 1 U Taq
polymerase. The PCR conditions used were as follows: a
denaturing step of 94°C for 3 minutes and then 35 cycles of
94°C for 15 s, 60°C for 15 s and 72°C for 30 s, with a final
extension at 72°C for five minutes. The PCR product was ana-
lyzed by electrophoresis in 4% Nu Sieve agarose gel (Master
Diagnostica) and visualized by ethidium bromide staining. An
undigested single band at 139 bp (when 20 CA repeats are
present) identified individuals who were AA homozygous, two
bands at 101 and 38 bp identified those who were GG
homozygous, and three bands at 139, 101 and 38 bp indi-
cated a heterozygote at the -1082 locus.
Statistical analysis
The main outcome measure was in-hospital mortality from any
cause, but we also assessed 90-day mortality. Sample size
was calculated considering a difference of 10% in hospital
mortality between groups as relevant, the frequency of the alle-
les in the population (control group), and a β error of 20% and
an α error of 5%. An interim analysis was planned after 225
patients were enrolled. At that point, the absolute difference in
mortality between AA individuals of TNF-β (the polymorphisms
more consistently associated with mortality) and GG/GA indi-
viduals was only 5%, and this was considered not clinically rel-
evant in septic patients, whose outcome is influenced by many
variables. With these data, it would be required to enroll 2,500
patients to achieve statistical significance. For these reasons,
we elected to terminate the study.
Descriptive results of continuous variables are expressed as
median (25th and 75th percentiles). The association between
risk factors and death was first examined by means of bivariate
analysis. This was accomplished using two-sample unpaired t-

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test for continuous variables after correction for equality of var-
iance (Levene's test), χ2 test, or Fisher's exact test for categor-
ical variables. Relative risks and their corresponding 95%
confidence intervals (CIs) were calculated. P < 0.05 was con-
sidered to reflect statistical significance.
A stratified analysis was performed before the multivariate
analysis using the Mantel-Hanszel χ2 test, in order to evaluate
the presence of interactions and confounding factors among
variables. A multivariate analysis using logistic regression anal-
ysis was used to determine variables independently associ-
ated with mortality in the entire group and only in those
patients who fulfilled septic shock criteria. The model was con-
structed using a forward stepwise method with the likelihood
ratio test. The variables tested for inclusion in the model had
an entry level P < 0.10 in the univariate analysis, but only those
with P < 0.05 are reported. The odds ratios (ORs) and their
corresponding 95% CI for each variable were also calculated
[21]. Moreover, in order to assess the associations among
quantitative variables with delayed initiation of adequate anti-
biotic therapy, a multiple linear regression analysis was per-
formed.
Results
A total of 293 patients were screened for inclusion in the
study, although 69 were excluded from the final analysis.
Therefore, only 224 patients were evaluable. The causes of
exclusion are listed in Table 1. Forty-six patients were hospital-
ized in the general ward and 178 patients were admitted to the
ICU (four of them were initially transferred to the general
ward). Fifty-two patients died in the hospital (23.2%), whereas
in-hospital mortality of the 69 excluded patients was 21.7% (P
> 0.05). The demographic data and the distribution of geno-
types of the 224 patients and 101 control individuals are sum-
marized in Table 2. The distribution of genotypes for the
analyzed polymorphisms did not differ between patients with
sepsis and control individuals.
At admission to the hospital, a clinical picture of sepsis was
present in 78 cases (34.8%), severe sepsis in 85 (38%) and
Table 1
Reasons for exclusion of 69 patients screened in the present
study
Reason Number
Permission denied 20
Diagnosis other than sepsis20
Race other than Caucasian 13
Do not resuscitate order8
Technical problem to extract DNA2
Death before permission could be obtained3
Other3
Table 2
Demographic data and genotype frequencies of TNF and IL-10 polymorphisms in patients with sepsis and controls.
Patients (n = 224)Control individuals (n = 101)
Age63.5 (49–72.75)50 (46.5–55.5)
APACHE II 14 (9–19)-
SOFA (1)a
4 (2–9)-
-308 TNF-α promoter polymorphism
GG186 (83) 82 (81.2)
GA 35 (15.6)15 (14.9)
AA 3 (1.4)4 (3.9)
TNF-β (NcoI polymorphism)
GG 16 (7.1)10 (9.9)
GA 69 (30.8) 34 (33.7)
AA 139 (62.5)57 (56.4)
IL-10-1082
GG 33 (14.8)15 (14.8)
GA 99 (44.2) 50 (49.5)
AA 92 (41)36 (35.7)
Values are expressed as median (25th to 75th percentile) or as n (%).aSOFA (1) means SOFA score of the first 24 hours in the hospital.
APACHE, Acute Physiology and Chronic Health Evaluation score; SOFA, Sequential Organ Failure Assessment; TNF, tumour necrosis factor.

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septic shock in 61 (27.2%). Twenty patients who were admit-
ted with sepsis developed severe sepsis or septic shock, and
37 patients with severe sepsis developed septic shock. There-
fore, 58 patients fulfilled only sepsis criteria, 52 patients pre-
sented with severe sepsis criteria and 114 patients presented
with septic shock. Only 10 patients received activated protein
C as part of their treatment.
All patients had clinical signs of infection but the causal micro-
organism could not be microbiologically documented in 66
patients (29.4%). Thirty-one patients presented with positive
blood culture and focus of infection, 25 were only bacterae-
mic, and in 102 only culture of material from the apparent
focus of infection was positive. Polymicrobial sepsis was diag-
nosed in 22 cases. Table 3 shows the micro-organisms iso-
lated in blood and the sites of infection.
Empirical antibiotic therapy was inadequate in 16 patients (in-
hospital mortality 75%). The reasons for inadequacy of antibi-
otic therapy were as follows: pathogen resistant to prescribed
antibiotic (eight cases), pathogen not covered by empirical
antibiotic therapy (four cases) and no antimicrobial adminis-
tered within the first 24 hours in the hospital (four cases). In
one case (bacteraemia of undetermined source caused by
Prevotella oris) the patient died before they received adequate
antimicrobial treatment.
Predictors of in-hospital mortality
A bivariate analysis of risk factors for in-hospital mortality is
reported in Table 4. The mortality rate was significantly higher
for females than for males. Neither microbiological documen-
tation of sepsis nor the presence of bacteraemia was related
to a worse outcome. Median delay of initiation of adequate
antibiotic therapy was significantly longer in nonsurvivors than
in survivors (the patient who died before receiving adequate
antimicrobial treatment was excluded from this analysis).
All genotype frequencies were in Hardy-Weinberg equilibrium.
There were no significant differences in genotype or allele fre-
quencies between survivors and nonsurvivors. A strong asso-
Table 3
Micro-organisms isolated in different sites of infections and bloodstream
Micro-organism Site of infectionBlooda
Escherichia coli
54 (35.3)27 (43.5)
Streptococcus pneumoniae
23 (15) 7 (11.3)
Streptococcus spp.15 (9.8) 8 (5.2)
Klebsiella spp.12 (7.8)6 (9.7)
Proteus mirabilis
7 (4.6) 2 (3.2)
Legionella pneumophila
7 (4.6)-
Enterococcus spp.6 (3.9)1 (1.6)
Pseudomonas spp 5 (3.3)-
Staphylococcus aureus
4 (2.6) 4 (6.4)
Neisseria meningitidis
4 (2.6) 3 (4.8)
Bacteroides fragilis
4 (2.6)2 (3.2)
Chlamydia pneumoniae
3 (1.9)-
Salmonella enteritidis
2 (1.3)1 (1.6)
Mycoplasma pneumoniae
2 (1.3)
Prevotella oris
1 (1.6)
Aeromonas hydrophila
1 (1.6)
Enterobacter spp.1 (0.65)
Nocardia asteroids
1 (0.65)
Staphylococcus spp. 1 (0.65)
Leptospira
1 (0.65)
Coxiella burnetii
1 (0.65)
Total 153 (100) 63 (100)
Values are expressed as n (%). aSix episodes of polymicrobial bacteraemia were detected.