Study setting and patients
This prospective observational study (ClinicalTrials.gov identifier: NCT03136081) was performed in two adult ICUs of a University hospital from June 2014 to May 2015. The study was approved by the ethic committee (Comité de Protection des Personnes Sud-Mediterranée III, 2014-A00500-47) and followed the STARD guidelines [15]. In accordance with French law, informed consent was obtained by the patient or his/her next of kin. Were included all consecutive adult patients admitted to the 2 participating ICUs with non-fungal septic shock or adult patients who developed septic shock during the ICU stay. Non-inclusion criteria were neutropenia (defined as a total leukocytes count < 500/mm3), immunosuppressive therapy, cancer related chemotherapy in the last year, history of bone marrow or solid-organ transplantation. The patients were then split into two groups (ICI and No-ICI, NICI) according to the occurrence of ICI during the ICU stay.
Definitions
Septic shock was defined by evidence of infection and a systemic response to infection, in addition to a systolic blood pressure of < 90 mmHg, despite adequate fluid replacement, and a need for vasopressors for at least 1 hour, according to the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee criteria [16]. The diagnosis of ICI was made based on the revised consensus definitions of invasive fungal infections developed by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Disease Mycoses Study Group [17]. ICI was defined by culture positives for Candida spp. from blood, per-operative peritoneal fluid or another sterile site. NICI was defined by the absence of proved ICI.
Baseline assessment and clinical data collection
Clinical data were recorded at ICU admission: demographic characteristics, severity of underlying medical condition according to simplified acute physiology score II (SAPS II) [18], Sepsis-related Organ Failure Assessment (SOFA) score [19], the presence of comorbidities, reason for admission to the ICU and cause of septic shock. During the ICU stay, the following data were collected at days 0, 2 and 7; SOFA score, length of stay, duration of mechanical ventilation, need for vasopressor and survival at ICU discharge. Antibacterial and antifungal drugs were given according to the recommendation applied in a same way in both ICUs.
Immunological data
EDTA-anti-coagulated tubes were collected on each visit day (days 0, 2 and 7) for blood cell count, lymphocyte phenotype, CD4 T-cell, CD8 T-cell count and mHLA-DR. Circulating mHLA-DR expression was assessed by flow cytometry (NAVIOS®; Beckman-Coulter) in accordance with the standardized recommendation [20, 21]. Monocytes were characterized on the basis of their CD14 expression. Results were expressed as the number of anti-HLA-DR antibodies per cell (AB/c).
Mycological data
Mycological samples were collected in case of infection suspicion according to the physicians in charge of the patient. Candida colonization was screened at days 0 and 7 in at least 3 sites from urine, gastric aspiration, tracheal aspiration and cutaneous swab. Candida Score [22] was calculated for each visit day. Colonization was defined when one nonsterile sample site was positive for Candida sp. Multifocal colonization was defined when more than one non sterile sample site was positive for Candida sp. Standardized procedures for in vitro identification of microorganism were used according to the usual procedures of the local mycology laboratory.
Mycological biomarkers
Β-d-glucan (BDG) was obtained on days 0, 2 and 7. Blood samples (15 mL) were centrifuged, separated into aliquots, and stored at -80 °C until analysis performed at the end of the study. The BDG assay (Fungitell®, Associates of Cap Cod Inc., Easy Falmouth, MA, USA) was performed according to the manufacturer’s recommendations. The cutoff value was set according to the company recommendations at 80 pg/mL [23, 24]. None of the results was available to the physician in charge.
Endpoints
The primary endpoint was the comparison of the kinetics during the first week after septic shock of mHLA-DR alone and in combination with BDG in ICI and NICI patients.
Statistical analysis
Data are expressed as mean ± SD or SEM for normally distributed data, and median with 95% confidence index (95%CI) for non-normally distributed data. Comparisons and biomarkers kinetics between ICI and NICI patients were made at day 0, 2 and 7. Continuous variables were compared using Student’s t test for normally distributed variables and the Mann-Whitney rank-sum test for non-normally distributed variables. The chi-square test or the Fisher exact test was used to compare categorical variables.
A cause-specific hazard model was built to assess the association of candida-related and immunologicals variables on the probability of death in the ICU or invasive candida infection [25]. In this model, the occurrence of ICI was the variable of interest, while death was considered as a competing event. Being discharged alive from the ICU was considered as a censored variable. Considering the low occurrence of candida infection only univariate analysis was performed. Candida variables and impaired immune function variables were fitted as time-dependent covariables. The direct effect on the risk of ICI was estimated by cause-specific hazard ratio (csHR).
Analysis of sensitivity, specificity, positive predictive value and negative predictive value was calculated for mycological and immunological markers separately and in association. For each marker, value over/under the threshold was only considered if before an event (ICI, death or ICU discharge). Delay of infection was estimated by the Kaplan-Meier method and compared between high and low risk patients (according to mHLA-DR and BDG values) with the log-rank test. A Monte Carlo simulation (considering the covariance between the two slopes) was also performed in order to assess the potentially best ROC curve. Expecting a 20% incidence of Candida sp positive samples following septic shock, a drop of 50% in mHLA-DR in the ICI patients by day 7 and no significant difference between day 0 and day 7 in NICI patients, we estimated that 50 patients with septic shock would be needed assuming a bilateral test, an alpha risk of 0.05 and a power of 0.8. A P value of less than 0.05 was taken as the significance level. We used SAS 9.4 (Sas Institute, NC, USA), R 3.0.2 (Vienna, Austria) and GraphPad Prism 6 for all the statistical analyses.