This prospective, observational, single-center study was conducted in the observation period from 03/2017 until 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 values 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 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 laboratory parameters (subsumed under the SOFA-Score, TISS-28, SAPS II)
- vital signs (heart rate, blood pressure, respiratory rate)
For characterization of the 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. Among other things, 23 different items such as the lungs, the cardiovascular system, frequency and extent of dressing changes, the kidney, the CNS, etc. are assigned to a certain point value. 5 of these items are additionally graded in their intensity (32). Ultimately, both scoring systems serve to make the severity of the illness of critically ill patients measurable and thus comparable 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 SOFA-Score inflammatory parameters (CRP, PCT, white blood cells) were collected.
Definition of delirium and its differential diagnoses - 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 (30). In the present study, the 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. Delirium was primarily diagnosed in all patients with a positive CAM-ICU test result, regardless of the presence of sepsis.
Definition of cognitive dysfunction
For example, critically ill patients with a limited vigilance level (RASS < -3) cannot be evaluated with current delirium screening instruments. Those patients cannot comply with simple prompts despite an appropriate sedation break and a RASS value greater than minus 3. Typical requests include 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 (29).
Definition of septic associated encephalopathy
Delirious conditions often occur in septic patients and can be the clinical manifestation of septic-associated encephalopathy. It is important to note that in particular structural changes of the brain due to craniocerebral trauma or ischemia as well as adverse drug reactions must be excluded before an SAE can be diagnosed (31,32). Validated delirium screening tools like the CAM-ICU have proven to be suitable for diagnosing SAE (33). 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 performed, for example due to cerebral damage, should not be classified in 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.
AChE-activity measurements
Since acetylcholine cannot be measured directly due to its rapid enzymatic degradation by acetylcholinesterase, it is necessary to define an appropriate surrogate parameter for the (central) cholinergic acetylcholine metabolism. The erythrocytic acetylcholinesterase activity (AChE-activity) has proven to be a suitable surrogate parameter in numerous studies (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 (28). 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 suitable (29). It is therefore useful to use the reference range of AChE-activity under clinical aspects as a rough guide. However, it should not be interpreted in a purely dogmatic way. Nevertheless, the majority of studies refer to these postulated reference ranges (26.7-50.9 U/gHb) when interpreting AChE-activity. However, it is often ignored that the underlying basis of these reference values are studies on healthy agricultural workers (34,35). Studies on the re-evaluation of reference values of AChE-activity in intensive care patients are still missing. In particular, inter- and intraindividual variability as well as time-dependent changes in AChE-activity must be considered when interpreting corresponding study results in critically ill patients.
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
With reference to previously published study results on AChE-activity, one of the main considerations in determining the number of cases was that, if available, statistically significant differences between non-deliriant and deliriant intensive care patients could be detected even in small case numbers. Further considerations for determining the sample size were based on the following facts:
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. Based on these facts, the number of cases was planned with around 200 patients in GPower 3.1. 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 patients 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 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 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.