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 × 109/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 × 104/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) . 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 . 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) . 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 . Side and forward scatter light are used to determine the intracellular structure and size of blood cells . 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.
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 . 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/μL), leukopenia (<4,000/μL), 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.
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 (X
2) 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 . 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.