The prospective, observational, single-center study was conducted in the observation period from 03/2017 until 03/2018 (positive vote of the local ethics committee, Trial-Code No. 363/16) in the interdisciplinary surgical intensive care unit (ICU), University Hospital of Ulm, Germany. The data evaluation took place in the period from 05/2018 to 06/2019. The study was retrospectively registered at the German Clinical Trials Register (DRKS-ID: DRKS00020542). The study protocol was in accordance to the Declaration of Helsinki ethical guidelines. All patients or their legal designees signed written informed consent to take part in this study.
Inclusion criteria:
- Age ≥ 18 years
- need for intensive care treatment due to an emergency or elective surgery
- expected stay on the ICU for at least 24 hours
- ≥ 2 values of AChE-activity
- ability to understand and speak the German language
Exclusion criteria:
- Age < 18 years
- < 2 consecutive measurement values of the AChE-activity
- missing informed consent
The following patient-related data were collected during the stay on the ICU:
- Age at enrollment
- gender
- ICU length of stay
- disease severity scores (SAPS II, TISS-28)
- Richmond Agitation Sedation Scale (RASS)
- Confusion assessment method for the intensive care unit (CAM-ICU)
- primary reason for ICU admission
- several laboratory parameters (subsumed under the SOFA-Score, TISS-28, SAPS II)
- vital signs (heart rate, blood pressure, respiratory rate)
For characterization of the heterogenous study population the relevant baseline data (demographic data, primary reason for ICU admission) were collected. The severity of illness was quantified using the Simplified Acute Physiology Score (SAPS II), as well as the Therapeutic Intervention Scoring System (TISS-28). The TISS-28 records the daily condition of the patient by recording the therapeutic, diagnostic and nursing measures (32). Both scoring systems display the severity of illness in critically ill patients and thus allow comparison within the framework of studies.
Definition of sepsis
Sepsis and septic shock respectively were diagnosed according to the third international consensus definitions for sepsis and septic shock (Sepsis-3) in 2016 (2). Patients were classified as septic if they met the criteria of the Sepsis-3 definition at admission or within 24 hours after admission to the intensive care unit (1,2). Beside the sequential organ failure assessment-Score (SOFA-Score), inflammatory parameters (C-reactive Protein (CRP), Procalcitonin (PCT), white blood cells) were collected.
Definition of delirium, cognitive dysfunction and septic associated encephalopathy
Definition of delirium
Delirium is a common syndrome on ICU and will be divided into the hypoactive-, the hyperactive- and the mixed- type (29). In the present study, the delirium was diagnosed by using the CAM-ICU, German Version. It was performed by trained personal (nurses and physicians) at least once every eight hours. Delirium was primarily diagnosed in all patients with a positive CAM-ICU test result, regardless of the presence of sepsis.
Definition of cognitive dysfunction
Critically ill patients with a lower vigilance level RASS < -3 cannot be examined with current delirium screening instruments such as the CAM-ICU. Those patients cannot comply simple prompts such as show one's teeth and tongue, squeezing one's hand e.g. These patients may also show up by an uncoordinated adaption to the respirator, agitation and the inability to reach a sufficient level of contact. The limitations mentioned for performing the CAM-ICU are often observed in critically ill patients with intracranial bleeding or neurocognitive disorders. For these patients the somewhat controversial term "cognitive dysfunction" has been chosen, which should be interpreted in a purely descriptive manner (28).
Definition of septic associated encephalopathy
In septic patients with delirious symptoms (CAM-ICU positive) septic-associated encephalopathy must be considered for differential diagnosis. However, before SAE can be diagnosed, structural changes of the brain due to craniocerebral trauma or ischemia as well as adverse drug reactions must be excluded (30,31). Validated delirium screening tools like the CAM-ICU have proven to be suitable for diagnosing SAE (32). Aware that the CAM-ICU can support the suspected diagnosis of SAE, but cannot prove it, the following consideration should be taken into account: Septic patients in whom the CAM-ICU cannot be reliably determined should not be assigned to the category "SAE". These patients are referred to as septic patients with cognitive dysfunction. Patients in the present study were suspected to have SAE under the following criteria: Diagnosed sepsis with concomitant delirious symptoms and positive CAM-ICU test result.
Cognitive dysfunction and septic-associated encephalopathy are often summarized under the generic term delirium. However, they are different from each other in significant aspects.
AChE-activity measurements
Since acetylcholine cannot be measured directly due to its rapid enzymatic degradation by acetylcholinesterase, an appropriate surrogate parameter for the (central) cholinergic acetylcholine metabolism is necessary. The erythrocytic acetylcholinesterase activity has proven to be a suitable surrogate parameter in numerous studies (27,28,33). One ethylenediamine tetraacetic acid (EDTA)-Blood sample (1 ml) was collected once daily over a period of maximum six days at 5:00 a.m. The first blood sample was taken in the morning after admission on the ICU, labeled as “day 1”. Between 7:00 and 12:00 a.m. the erythrocyte AChE-activity was determined by using LISA-ChE® (Dr. F. Koehler Chemie GmbH, Germany), a point-of-care testing device. The measurement of the AChE-activity is based on the modified Ellman method, a colorimetric method, improved by Worek et al (27). The reference values of AChE-activity ranges from 26.7 U/gHb until 50.9 U/gHb (27,28). Studies on the re-evaluation of reference values of AChE-activity in intensive care patients are still missing up to now. Moreover, the inter- and intraindividual variability and the time-dependent changes in AChE-activity in critically ill patients have not yet been adequately investigated. The primary endpoint of this study was to investigate whether AChE-activity is altered in septic patients with suspected SAE compared to non-septic patients with and without delirium. The secondary endpoint was to investigate whether AChE-activity is capable of differentiating between SAE and other causes of delirium in critically ill patients.
Sample size calculation and power analysis
Previously published data on AChE-activity in delirious intensive care patients have shown that even in small case numbers a statistically significant change in AChE-activity can be detected (28,33). Own data were used as a pilot data set for the sample size planning of this study. In addition, the following considerations were taken into account: the average number of intensive care patients in the interdisciplinary surgical intensive care unit, University hospital Ulm, is about 550 patients per year. The prevalence of sepsis in German intensive care units was about 12.4% (sepsis) and 11.0% (severe sepsis and septic shock) in the observation period. A total number of 200 patients was calculated for the prospective observational study (GPower 3.1). After completion of the patient recruitment, which was limited for a maximum period of 12 months in the study plan, the patients were divided into a septic and a non-septic group. Subsequently, a post-hoc power calculation was performed. In detail, a simulation-based approach has been used in order to assess the power associated to a longitudinal AChE-activity regression model including the time point of measurement, group status of the patient (septic vs. non-septic) as well as the corresponding interaction term. This analysis was conducted by means of the SIMR package in R (version 3.6.1). Based on the sample size of about 40 patients in the smaller (septic) subgroup, the simulation shows an empirical significance of about 60%. Considering the statistically significant results, the empirical power is calculated in a range between 60-80% despite the large difference.
Statistical analysis
Data were collected in Microsoft Excel 2010 (Microsoft Corp., Redmond, WA) and analyzed by using GraphPad PRISM, Version 5 for Windows and SAS Version 9.4.
AChE-activity was analyzed over the course of time by using a linear regression model accounting for repeated measures. The AChE-activity was defined as the dependent variable and the duration of measurement (a maximum of six consecutive days) was defined as the continuous independent predictor of primary interest. Using a time adjusted model, the effect of further possible predictors of AChE-activity was analyzed.
Quantitative data were expressed as median, minimum and maximum and, for nonparametric distributions, were compared using the Wilcoxon matched-pairs test. For the analysis of the independent samples, we used the Mann-Whitney test. All results reported have to be interpreted in an exploratory manner, since we did not adjust the p-values for multiple testing.