The intensive care infection score – a novel marker for the prediction of infection and its severity
Study design and patients
The ICIS study is an add-on non-interventional study of patients who had been enrolled
into a prospective, cluster-randomized, crossover trial, involving both intensive
care units (ICUs) of the Erasmus Medical Center Rotterdam and both ICUs of the Maasstad
hospital Rotterdam. The ICUs were stratified and randomized by treatment regimen into
a control group (standard of care) and an intervention group. In the intervention
arm, blood culturing for a suspected infection was guided by PCT measurements. The
acronym for PCT-guided blood culturing in the intensive care, ProBIC, was used for
this study and results will be reported later. The trial was conducted between January
2013 and September 2014. The ICU of the Erasmus Medical Center is a tertiary care
mixed medical-surgical ICU with approximately 2000 admissions per year. The ICU of
the Maasstad hospital is a secondary care mixed medical-surgical ICU with 1200 admissions
per year.
The trial was conducted in accordance with the ethical principles decreed by the Declaration
of Helsinki and in compliance with International Conference on Harmonization Good
Clinical Practice Guidelines. The institutional review board (IRB) or the independent
medical ethical committee at each of the investigational centers (Medisch Ethische
commissie Maasstad ziekenhuis, Rotterdam, Nederland and Medisch Ethische commissie
Erasmus Medisch Centrum, Rotterdam, Nederland) reviewed and approved the protocol,
amendments and informed consent document. The medical ethical committee of the Erasmus
Medical Center finally approved the study (MEC 2011-505). The trial was registered
at ClinicalTrial.gov (protocol ID NCT01847079) on 24 April 2013. All patients or their
proxy provided written informed consent prior to study inclusion, at ICU admission.
Inclusion criteria were age above 18 and below 80 years and the clinical suspicion
of infection, for which the attending intensivist established a medical need for blood
culture. Suspicion of infection included but was not limited to increased body temperature
above 38.3 °C (tympanic temperature), chills, progressive leukocytosis, increased
CRP, increasing consolidation on chest radiography or other imaging of potential infection
sources. It was possible for each patient to be included more than once, but in the
current study we only analyzed the first time that blood was sampled for culture.
Patients were excluded if they were pregnant, had neutropenia (defined as leukocyte
count less than 0.5?×?10
9
/L), used immunosuppressive or immunostimulatory therapy, or had a predetermined illness
with death expected within 24 h. Patients were not included if blood cultures were
performed as part of a standard protocol (such as patients with veno-venous or veno-arterial
extracorporeal membrane oxygenation (ECMO)) or were performed to check the effectiveness
of treatment (such as in endocarditis), unless the blood culture was done because
of suspicion of infection. The ICUs switched the allocated regimen every 3 months,
so that there were six 3-month episodes of standard care in which 774 patients were
eligible for inclusion, and 473 patients were excluded (5 patients who were ?18 years
of age; 63 with neutropenia (0.5?×?10
4/
L); 35 with uncontrolled malignancy; 256 on immunosuppressive medication; 22 who were
expected to die within 24 h; and 92 without informed consent). Data for the ICIS study
were thus collected in 301 patients in the control arm (six 3-month episodes) of the
ProBIC study.
Study protocol, data collection and assays
Baseline demographic data and clinical variables were recorded on the day of inclusion,
and included age, sex, comorbidity, reasons for admission, use of antibiotics including
selective decontamination of the digestive tract (SDD), antifungal treatment, steroids,
immunosuppressive medication, immune status and recent surgery. The treatment received
during ICU stay was also recorded and included mechanical ventilation, renal replacement
therapy, total parenteral nutrition, arterial and central venous catheters, and the
use of vasopressor or inotropic medication. The acute physiology and chronic health
evaluation II (APACHE II) and the sequential organ failure assessment (SOFA) score
were recorded at admission. The length of ICU and hospital stay and vital outcomes
were recorded for up to 90 days after inclusion.
At the same time that blood was taken for culture, blood samples were taken for determination
of WBC, CRP, PCT, and ICIS (day 0). Blood for similar measurements (except for PCT)
was taken in the morning on the two following days (days 1 and 2). Treating physicians
and investigators were blinded to the PCT and ICIS measurement results. Also the outcome
adjudicators that decided presence or absence of infection were blinded to the biomarker
results. Two sets of blood cultures were taken and directly sent to the department
of medical microbiology. The set taken for blood culture consisted of one aerobic
and one anaerobic bottle (BD Bactec™, Franklin Lakes, NJ, USA), which contain resin
to enhance recovery of organisms. The samples were incubated for a 7-day period in
an automatic analyzer (BD Bactec™) that automatically demonstrates the time to positive
blood culture in the case of positive bacterial or fungal growth. Gram strains were
performed, and the organisms were cultured on agar plates after identification of
growth using the VITEK® 2 (Biomerieux, Marcy l’Etoile, France).
Blood for the WBC and ICIS measurement was obtained in a K3EDTA tube. Both the WBC
and ICIS parameters were measured on a modified fluorescence flow hematology analyzer
with fully automated gating (Sysmex, Kobe, Japan) 14]. The ICIS was measured promptly after collection but within a maximum of 24 h. The
ICIS score is composed of five blood-cell-derived parameters that characterize the
innate immune response 15]–19]. The five parameters include the mean fluorescence intensity of mature (segmented)
neutrophils, the difference in hemoglobin concentration between newly formed and mature
red blood cells, the total segmented neutrophil count, the antibody secreting lymphocytes,
and the accurate immature granulocytes count, as previously described 12]. Each parameter is available from a standard routine method and can be measured within
1 minute without sample preparation on a modified fluorescence flow hematology analyzer
with fully automated gating (Symex) 12]. The methodology is based on routine hematology fluorescence flow cytometry using
different fluorescence reagents for mainly nucleic acids, and specifically designed
blood cell membrane surfactant reagents generating information about cell shape and
the formation of bioactive lipids from cell membranes 12]. Side and forward scatter light are used to determine the intracellular structure
and size of blood cells 12]. By adding all weighting values for all five parameter components, the maximum possible
ICIS is 20. Serum CRP (turbidimetric assay) and PCT (electrochemiluminescence BRAHMS
immunoassay) measurements were routinely performed using a Cobas 8000 platform (Roche,
Almere, Netherlands). Blood for PCT measurement was sampled in a z serum clot activator
tube.
Definitions
After completion of the study, the investigators decided whether an infection was
present from days 0–2, on the basis of the available imaging and culture results.
The outcome adjudicators were blinded to all biomarkers. Source and likelihood of
infection were based on criteria defined at the International Sepsis Forum Consensus
Conference 20]. Culture results were analyzed within a 48-h window from before and after taking
blood cultures. The causative microorganisms were recorded. BSI was defined as a positive
blood culture with a recognized pathogen except for skin contaminants 20], 21]. In the case of skin contaminants, BSI was identified if at least two blood cultures
drawn on separate locations were positive 20], 21]. Patients were divided into groups according to increasing likelihood of infection
and invasiveness of associated microorganisms that was suggestive of increasing severity:
group 1 without infection or with possible infection irrespective of cultures; group
2 with probable (irrespective of cultures) or proven local infection (with positive
cultures of a causative microorganism) without BSI; and group 3 with BSI irrespective
of local infection. SIRS was defined as two or more of the following criteria: (1)
body temperature 38 °C or 36 °C; (2) WBC (10,000/?L), leukopenia (4,000/?L), or
10 % bands; (3) heart rate 90 beats/minute; and (4) respiratory rate 20 breaths/minute
or mechanical ventilation, for values at day 0. When SIRS and a probable/proven infection
(groups 2 or 3) were present, patients were classified as having sepsis. Shock was
defined as acute circulatory failure characterized by persistent systolic arterial
pressure 90 mm Hg or mean arterial pressure (MAP) 70 mm Hg for at least 1 h despite
adequate fluid resuscitation or requirement of vasopressor support to maintain MAP,
at day 0. In the presence of sepsis, shock was defined as septic shock.
Statistical analysis
This was performed using SPSS version 23 (SPSS inc., Chicago IL, USA) and using R
package. Data are expressed as median (interquartile range) or as number of patients
(percentage) where appropriate. Most data were distributed non-normally (Kolmogorov-Smirnov
test P??0.05). Group (2) differences were evaluated using the Kruskal-Wallis test or chi-square
(X2
) test, for continuous and categorical data, respectively. The Mann-Whitney U test and Fisher exact test were used to compare two groups. To evaluate predictive
values we calculated the areas under the receiver operating characteristic curves
(AUROC) for day 0 values. For the predictive values of sepsis and septic shock we
used the values for day 0. We consider an AUROC 0.70 as clinically relevant 22]. The optimum cutoff value was calculated on the basis of the highest sensitivity
and specificity combined (Youden index). Positive and negative predictive values were
calculated. To correct for multiple testing we set the level of statistical evidence
at P???0.01. Exact P values 0.001 are given.