This prospective, observational, single-center study was conducted between 03/2017 and 03/2018 after the positive ethics committee vote of the University of Ulm (Trial-Code No. 363/16). 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 conformed 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 were as follows:
- 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 readings of AChE-activity
- ability to understand and speak the German language.
Exclusion criteria were as follows:
- Age < 18 years
- < 2 consecutive measurement values of the AChE-activity
- missing informed consent
The patients were not followed up after discharge of the ICU.
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)
- primary reason for ICU admission
- several laboratary parameters
During the observation period 241 patients were admitted to the surgical ICU of the University hospital Ulm, Germany which had been considered potentially eligible. However, 66 patients did not meet inclusion criteria. In detail, for 38 patients, the number of AChE-activity was less than 2 readings (length of stay on ICU < 24 hours: 10 patients deceased, 28 patients were soon relocated to the Intermediate Care Unit), 10 patients had incomplete data, 18 patients revealed a missing written consent at the time of data evaluation. Finally, a total of 175 patients were included in the further analysis. After admission to the ICU patients were divided into non-septic and septic and were screened for delirium or cognitive dysfunction (for details see below), respectively, every 8 hours.
130 patients were classified as non-septic with a median age of 64 years at enrollment. 89 of them were men, 41 of them women. The median length of stay on the ICU was 8 days. A total of 10 non-septic patients deceased during the observation period. 36 of the non-septic patients were CAM-ICU positive, i.e. they had a delirium. 12 of them exhibited a hypoactive, 5 a hyperactive and 19 a mixed-form as delirium subtype. As a differential diagnosis of delirium, 24 of the 130 non-septic patients were classified as cognitive dysfunctional.
45 of 175 patients revealed a sepsis. 40 of these patients were CAM-ICU positive, in whom a septic associated encephalopathy was considered as a differential diagnosis. One patient was cognitively disturbed due to cerebral hemorrhage, three patients were sedated until death and at least one patient was permanently CAM-ICU negative.
The median age in the septic group was 61 years. The gender distribution in the group of the septic patients showed 33 male and 12 female patients. The median length of stay in the ICU was 14 days for the septic subgroup. 22 of the septic patients deceased during the observation period.
Subgroups for patients with delirium and altered AChE-activity were built dependent if an increase or decrease of AChE-activity was observed defined as a 10% change of the individual baseline value. For study population characterization relevant baseline data (demographic data, primary reason for ICU admission) were collected; severity of illness was estimated using Simplified Acute Physiology Score (SAPS II), a scoring-system which was developed to calculate the physiologic condition of critically ill patients and the Therapeutic Intervention Scoring System (TISS-28), addressing the daily care and therapy effort.
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 into the sepsis group if they met the criteria on admission or within 24 hours after admission on ICU (1,2). Beside the SOFA-Score, inflammatory parameters (CRP, PCT, white blood cells) were collected.
Definition of delirium and its differential diagnoses of cognitive dysfunction and septic associated encephalopathy
Delirium is a common syndrome on ICU and is clinically divided into the hypoactive form , the hypoactive and the mixed form (32). Delirium was diagnosed by using the Confusion assessment method for the Intensive Care Unit (CAM-ICU), German Version. It was performed by trained personal (nurses and physicians) at least once every eight hours, or more often if considered necessary. If the patient was not able to perform basic executive functions on request (showing the teeth, stick out the tongue, squeeze the hand) despite an adequate sedation level (RASS better -3) or, if patients without sedation displaying an inadequate awakening reaction, e.g. pressing against mechanical ventilation, agitation without reaching a contact level, were classified with “cognitive dysfunction” (31). In this study, the term "cognitive dysfunction" was used as a differential diagnosis of ICU delirium considering the fact that the CAM-ICU has a ”diagnostic gap” if patients are cognitively impaired, e.g. due to cerebral hemorrhage, even though they meet the formal test criteria of the CAM-ICU such as a sufficient RASS level.
Septic patients with positive CAM-ICU were attributed to the differential diagnosis “septic associated encephalopathy (SAE)”. Primary infections of the central nervous system, structural alterations of the brain (due to craniocerebral trauma or ischemia) as well as adverse drug reactions have to be excluded per definition prior to the diagnosis of SAE (33,34). In absence of specific tests for the diagnosis of SAE, validated delirium-screening-tools, like the CAM-ICU, have proven to be suitable (35).
AChE-activity measurements
Since acetylcholine is not directly measurable due to the rapid enzymatic degradation by acetylcholinesterase, it is necessary to get access to a corresponding surrogate parameter for the (central) cholinergic acetylcholine metabolism. For monitoring the cholinergic functional state, the erythrocyte acetylcholinesterase-activity (AChE-activity) as a surrogate marker of the central nervous acetylcholine transmission, could be analyzed (33).
One EDTA-Blood sample (1 ml) was collected once daily over a period of maximum six days at 5:00 a.m. Several patients got an infusion of indirectly acting parasympathomimetics, usually at 6:00 a.m. (for intestinal stimulation, average half-life up to 80 minutes) which could suppress AChE-activity. 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), 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 (30). A relevant change of the AChE-activity was defined as an increase or decrease of the AChE-activity for at least 10%, referring to the first measured AChE-activity over the observation period. The literature based reference values of AChE-activity ranges from 26.7 U/gHb until 50.9 U/gHb (36,37). Due to a high inter- and intra-individual variability of AChE-activity, in clinical practice, a modified reference range from 30.0 to 50.0 U/gHb is more feasible (31,36). The above-mentioned reference ranges could therefore be mentioned as a rough guide in the interpretation of AChE-activity, but should not be understood as a dogma.
Primary endpoint of this study was to investigate, whether AChE-activity is altered in septic patients with SAE / delirium compared to non-septic patients with and without delirum. As a secondary endpoint, it was examined whether AChE-activity is suitable to differentiate between SAE and other delirium entities in critically ill patients.
Sample size calculation and power analysis
With reference to previously published study results on AChE-activity, it has been shown that, if available, statistically significant differences between non-delirant and delirant intensive care patients can be demonstrated even with small numbers of cases. Further considerations in sample size calculation were based on the following facts:
The average number of intensive care patients in the interdisciplinary surgical intensive care unit, University hospital Ulm, is on average about 550 patients per year. Experience has shown that approximately half of all admitted patients meet the inclusion- and exclusion criteria mentioned, which corresponds approximately to the number of eligible patients in the present study (n = 241). 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. Based on these facts, the number of cases was planned with around 200 patients. A post-hoc power calculation was done using the study data of septic and non-septic patients. Specifically, 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), which revealed that a number of about 100 patients per group (septic and non-septic, i.e. n = 200 patients in total) would be required to be assessed longitudinally in order to reach a statistical power of 80%. Based on the currently available sample size of about 40 patients in the smaller (septic) subgroup, the simulation reveals an empirical power of about 60%. Due to the large difference in cohort size (45 septic vs. 130 non-septic patients) the overall empirical power thus ranges somewhere between 60-80%.
Statistical analysis
Data were collected in Microsoft Excel 2010 (Microsoft Corp., Redmond, WA) and analysed by using GraphPad PRISM, Version 5 for Windows and SAS Version 9.4.
AChE-activity was analysed 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 time of measurement (a maximum of six consecutive days) was defined as the continuous independent predictor of primary interest.
By 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 Wilcoxon matched-pairs test. For the analysis of the independent samples, we used the Mann-Whitney test. All results reported shall be interpreted in an exploratory manner, since we did not adjust the p-values for multiple testing.