Sepsis Clinical Trial
Official title:
Bacterial DNA Detection as a Diagnostic Tool of Infection in Critical Ill Patients With SIRS
Sepsis is a common cause of morbidity and death in intensive care units. Clinical and
laboratory signs of systemic inflammation, including changes in body temperature,
tachycardia, or leukocytosis, are neither sensitive nor specific enough for the diagnosis of
sepsis. The diagnosis of sepsis is difficult, because clinical signs are unspecific. These
signs include tachycardia, leucocytosis, tachypnoea, and pyrexia, which are collectively
termed a systemic inflammatory response syndrome (SIRS). SIRS is very common in critically
ill patients, being found in various conditions including trauma, surgery, burns,
pancreatitis, post-cardiac arrest syndrome, cardiac surgery. Microbiological culture can be
used to distinguish sepsis from non-infectious conditions. However, this method lacks
sensitivity and specificity, and there is often a substantial time delay. So these signs can
also be misleading because critically ill patients often present with the systemic
inflammatory response syndrome without infection. This issue is of paramount importance,
since therapy and outcome differ greatly between patients with and those without sepsis;
clinicians are often prone to overuse antibiotic therapy being afraid of not treating a
potential infection or superinfection. Moreover, the widespread use of antibiotics for all
such patients is likely to increase antibiotic resistance, toxicity, and costs. On the
opposite, any delay in administration of antibiotics can be extremely detrimental for the
infected patient with an exponential increase of the odd ratio for death. Search for early
biomarker tools for the diagnosis of infection, initially promising, are quite challenged
and controversial nowadays because they can be more related to the inflammation response,
irrespective to the insult. Furthermore up to 40% of the infections remain strongly
suspected but not bacteriologically documented. Persisting researches are ongoing to find
new markers to better discriminate SIRS related to infection process from to SIRS not
related to infection. Cytokine profiles using multiplex analysis seems more related to the
severity of the SIRS than the trigger of the SIRS (infectious or non infectious diseases).
Thus, new tools have been developed to identify bacteria by detecting their DNA by various
techniques. These techniques have many potential interests over conventional microbiologic
tests by decreasing turnaround time (within a few hours 2-6 hours), reducing inhibitory
effects of prior use of antibiotics, detection of slow or fastidious growing organisms.
However these tests remain to be validated in a clinical setting.
The goal of the current study is to evaluate the diagnostic value of plasma detection of
bacterial DNA in ICU patients with a clinical suspicion of bacterial infection.
The goal of the current study is to evaluate the diagnostic value of plasma detection of
bacterial DNA in ICU patients with a clinical suspicion of bacterial infection.
Definitions of septic syndromes based from the ACCP/SCCM expert panel. Terms Definition
criteria Infection Invasion by microorganisms of an normally sterile tissue
Systemic inflammatory response Syndrome (SIRS) At least 2 of 4 following criteria:
- Temperature > 38°C or < 36°C
- Heart Rate > 90 beat/min
- Tachypnea > 20/min or PaCO2 < 32 mmHg
- WBC count > 12 000/mm3 or < 4 000/mm3 Sepsis SIRS related to an infectious process
Severe Sepsis Sepsis with :
- Arterial hypotension (SBP<90 mmHg, MAP<70 or SBP decrease < 40 mmHg)
- Or any other organ dysfunction :
- PaO2/FiO2 < 250 (or < 200 if pneumonia)
- Oliguria (< 0,5 ml/kg/h for at least 2 hours)
- Hyperlactacidemia ( > 3mmol/l)
- Platelet count < 80000/mm3 Septic shock Severe sepsis with persisting hypotension ( > 1
hour) despite adequate fluid resuscitation and requiring vasopressor support
(epinephrine or norepinephrine).
SBP: Systemic blood pressure, MAP: Mean arterial blood pressure,
All codes and definitions are established prior to study initiation. The following
information are recorded: age and sex, admission category (medical, scheduled surgery, or
unscheduled surgery), origin (home, ward, or emergency room), and McCabe score, ICU and
hospital mortality. Severity of illness was evaluated on the first ICU day using the
Simplified Acute Physiology Score (SAPS II), Sepsis-related Organ Failure Assessment (SOFA)
score, and Acute Physiologic and Chronic Health Evaluation (APACHE) II score. Knaus scale
definitions were used to record preexisting chronic organ failures including respiratory,
cardiac, hepatic, renal, and immune system failure.
The presence or absence of infections was documented according to the standard definitions
developed by the Centers for Disease Control; We will determine three groups: 1. patients
with documented infection 2. patients without documented infection and 3. patients with
suspected but non documented infection.
Study protocol Four centers will participate (65 beds overall). Along with clinical
C-reactive protein (CRP) and procalcitonin (PCT) measurements used in clinical routine lab
measurements, two blood samples (4.5 mL each) will be collected. CRP and PCT are collected
routinely when patient meet criteria for SIRS in all three ICUs to helps us distinguishing
infectious from non-infectious SIRS. One whole blood on EDTA will be rapidly processed for
bacterial/fungi DNA detection. (frozen until analysis or within 72 hours the sample kept at
4°C).
The second sample using tubes containing Gel and dipotassium EDTA allowing separation after
centrifugation (1500g for 10 minutes at 4°C) of the plasma from (white and red) blood cells
without any manipulation and thus lowering risk any nurses' occupational exposure. Then the
plasma will be stored at -80°C until analysis. Blood collection will be performed when SIRS
criteria will be met either at admission or later on during the ICU stay (suspicion of
surperinfection).
Bacterial and Fungal DNA detection The bacterial and Fungal DNA detection will be performed
using the Multiplex PCR pathogen detection VYOOTM (SIRS-Lab GmbH, Jena, Germany). VYOOâ
combines culture-independent pathogen-derived nucleic acids concentration and multiplex
PCR-based species detection. The multiplex PCR delivers results of high therapeutic value
within ~6 hours and detects 34 bacterial and 6 fungal species that cause life-threatening
infections as well as five most frequent resistance markers. In order to ensure highest
clinical validity, the primer selection of the species and the antibiotic resistances bases
on international study results on septic infections. The system was proved to be far less
susceptible for contaminations than blood cultures or alternative NAT protocols and ensures
increased specificity and sensitivity.
VYOOâ achieves its high sensitivity with the included sample preparation system LOOXSTER®
which bases on SIRS-Lab's proprietary PUREPROVE® technology. LOOXSTER® specifically
concentrates the minute quantity of bacterial and fungal DNA from a huge human DNA
background. Total DNA is applied onto an affinity chromatography column with a
matrix-immobilized DNA binding protein that recognizes definite motives within the pathogen
DNA. More than 90% of the human background DNA is removed. This effect substantially
increases the sensitivity of the downstream multiplex PCR protocol and simultaneously
reduces the time-to-result.
The pathogen cells within 5 ml whole blood are disrupted mechanically using glass beads
(0.1/2.5 mm in diameter) and a lysis device (e. g. FastPrep®-24, MP Biomedicals, Solon, OH,
USA). After a proteolytic digestion step, total DNA is isolated using a short spin column
protocol. The total DNA is dissolved in an appropriate buffer and applied to the LOOXSTER®
spin column. The final bacterial plus fungal/human DNA ratio is significantly increased and
the concentrated pathogen DNA is directly used for multiplex PCR. Generated pathogen
specific amplicons are visualized by gel electrophoresis and compared with VYOOâ-specific
DNA length markers for the proof of present pathogens or resistances. An alternative
amplicon attribution is achieved by hybridization (ATâ system, Clondiag), which ensures a
higher degree of specificity and sensitivity.
Cytokines detection by ELISA Dosage of plasma cytokines (IL-6, 8, 10) will be performed by
ELISA methods using commercially available kits (R&D systems, Minneapolis, MN) or by
Bio-Plex Multiplex Cytokine Assay (BioRad Lab., Hercules, CA).
Endotoxin & Peptidoglycan detection For endotoxin dosage, we used Limulus Amebocyte Lysate
(LAL) reagent water (LRW) (Biowhittaker) and non-pyrogenic pipettes and tips (Costar). The
samples will be diluted with LRW in non-pyrogenic 96-well culture plates (Costar). Blood
samples will be collected in endotoxin free tubes containing a gel that after centrifugation
makes a barrier between the cellular pellet and the plasma (Becton Dickinson, Franklin
Lakes, NJ). The plasma will be then transferred to non-pyrogenic tubes and kept at -20°C
until tested. Detection of bacterial endotoxin in plasma is hindered by its yellow
coloration that interferes with the Limulus amebocyte lysate test and gives an absorbance at
405 nm, but also by the presence of inhibitors. We overcame the background absorbance of
plasma using a Diazo-coupling Limulus amebocyte lysate assay that gives a magenta coloration
(Associates of Cape Cod Inc.). Furthermore, in order to inactivate the inhibitors, plasma
were diluted 1:4 in LRW and heated for 30 min at 60°C as it has been shown that this
dilution and heating procedure allow optimal endotoxin detection in plasma. After heating,
50 μl of diluted plasma were transferred in 96-well culture plate and incubated with the
same volume of Limulus amebocyte lysate for 30 min at 37°C. The reaction will be stopped, as
specified by the manufacturer, by successive addition of 50 μl of sodium nitrite in HCl, 50
μl of ammonium sulfamate and 50 μl of N-(1-naphthyl)-ethylenediamine (NEDA) that form a
diazotized magenta derivative. The optical density will be read at 570 nm and endotoxin
concentration will be determined by comparison with an endotoxin standard curve. The
detection limit of the test is 0.02 EU/ml of endotoxin.
Peptidoglycan will be measured according to a newly experimental procedure set up in Dr.
Cavaillon's laboratory. The technique that detects peptidoglycan from both Gram-positive and
Gram-negative bacteria has already been shown to detect peptidoglycan in plasma from
patients with sepsis. Because a patent has been applied no further technical information can
be presently provided in the present version but will be added as soon as possible.
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