Cholinesterase activities and sepsis-associated encephalopathy in viral versus nonviral sepsis

Abstract

Purpose

There is evidence that cholinergic imbalance secondary to neuroinflammation plays a role in the pathophysiology of sepsis-associated encephalopathy (SAE). Blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities have been proposed as surrogate parameters for the cholinergic function of the central nervous system. Viral sepsis is associated with systemic inflammation and BChE has been reported to be of prognostic value in a small cohort of COVID-19 patients. Nevertheless, the prognostic value of AChE in patients with viral sepsis remains unclear.

Methods

We investigated the role of AChE and BChE activities as prognostic biomarkers of SAE and mortality in patients with viral vs nonviral sepsis enrolled in two prospective cohort studies. We quantified the AChE and BChE activities in whole blood of patients at two time points in the acute phase of viral sepsis (N = 108) and compared them with the activities in patients with nonviral sepsis (N = 117) and healthy volunteers (N = 81). Patients were observed until discharge from the intensive care unit (ICU).

Results

Three days after sepsis onset, the median [interquartile range] levels of AChE and BChE were reduced in both patients with viral sepsis (AChE, 5,105 [4,010–6,250] U·L⁻¹; BChE, 1,943 [1,393–2,468] U·L⁻¹) and nonviral sepsis (AChE, 4,424 [3,630–5,055] U·L⁻¹; BChE, 1,095 [834–1,526] U·L⁻¹) compared with healthy volunteers (AChE, 6,693 [5,401–8,020] U·L⁻¹; BChE, 2,645 [2,198–3,478] U·L⁻¹). Patients with viral sepsis with SAE during their ICU stay had lower AChE activity three days after sepsis onset than patients without SAE (4,249 [3,798–5,351] U·L⁻¹ vs 5,544 [4,124–6,461] U·L⁻¹). Butyrylcholinesterase activity seven days after sepsis onset was lower in patients with viral sepsis who died in the ICU than in surviving patients (1,427 [865–2,181] U·L⁻¹ vs 2,122 [1,571–2,787] U·L⁻¹).

Conclusion

Cholinesterase activities may be relevant prognostic markers for the occurrence of SAE and mortality in the ICU in patients with viral sepsis.

Study registration

This study constitutes an analysis of data from the ongoing studies ICROS (NCT03620409, first submitted 15 May 2018) and ICROVID (DRKS00024162, first submitted 9 February 2021).

Supplementary Information

The online version contains supplementary material available at 10.1007/s12630-024-02692-7.

Sepsis-associated encephalopathy (SAE) is a frequent organ dysfunction in sepsis, associated with poor outcome.¹ Various descriptions of SAE exist, but all are commonly defined as a reversible global impairment of consciousness in patients with sepsis excluding other causes.² Neuroinflammation secondary to systemic inflammation is associated with an imbalance of the neurotransmitter acetylcholine (ACh) in the central nervous system (CNS).^3–5 This cholinergic imbalance has been proposed as a possible cause of SAE.⁴

In the peripheral nervous system, ACh modulates the host immune response via the cholinergic anti-inflammatory pathway.⁶ In homeostasis, inflammation sensed by the vagus nerve leads to the secretion of ACh via vagal nerve endings. Acetylcholine binds to nicotinic, α-bungarotoxin-sensitive receptors on macrophages and lymphocytes, inhibiting the synthesis of proinflammatory cytokines and counterbalancing the sensed inflammation.^6–10 A functional cholinergic anti-inflammatory pathway has been reported to be protective in experimental sepsis.¹¹

Cholinergic activity in the CNS can be evaluated by measuring the activities of the ACh-degrading enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in whole blood^12,13 as their activities in blood correlate with those in the CNS.^14–16 Acetylcholinesterase is present in the nervous system and in the cytosol of erythrocytes.¹⁷ Butyrylcholinesterase is produced in the liver and present in the plasma.¹⁸

The general objective of this study was to evaluate the prognostic value of AChE and BChE activities in patients with viral sepsis. Because of its lower prevalence compared with bacterial sepsis, viral sepsis has received less scientific attention as a separate sepsis entity despite calls for the adoption of a personalized approach to sepsis therapy.¹⁹ The COVID-19 pandemic, during which a persistent subset of patients required intensive care,²⁰ made the need for a better understanding of viral sepsis evident. Influenza is another common cause of viral sepsis. Severe COVID-19 and influenza are characterized by many common immunological abnormalities, including systemic hyperinflammation referred to as cytokine storm.²¹

Hitherto, cholinesterase activities have rarely been assessed in viral sepsis. A prospective cohort study of 54 patients with severe COVID-19 found reduced levels of BChE activity compared with a historical healthy control group of 40 volunteers.²² Acetylcholinesterase activity has not been assessed in viral sepsis. Because viral sepsis is associated with systemic inflammation, we hypothesized a cholinergic imbalance in patients with viral sepsis—as seen in reduced cholinesterase activities—and a prognostic relevance thereof. The specific aims of this study were to evaluate 1) the effects of viral sepsis vs nonviral sepsis on AChE and BChE activities, 2) the prognostic values of AChE and BChE activities for the occurrence of SAE, and 3) the prognostic values of AChE and BChE activities for intensive care unit (ICU) mortality. To address these aims, we conducted an interim analysis of two ongoing prospective sepsis cohort studies including > 300 critically ill and healthy volunteers.^23,24 We quantified AChE and BChE activities in patients at two time points in the acute phase of viral sepsis compared with nonviral sepsis and healthy volunteers.

Methods

Study design

This study constitutes a subanalysis of the ongoing prospective studies Identification of cardiovascular and molecular prognostic factors for the medium-term and long-term outcomes of sepsis (ICROS, NCT03620409)²³ and Identification of cardiovascular and molecular prognostic factors for the morbidity and mortality in COVID-19-sepsis (ICROVID, DRKS00024162),²⁴ in accordance with the Declaration of Helsinki. The Ethics Committee of the Friedrich Schiller University Jena (Jena, Germany) approved both studies (ICROS: 5276-09/17, 2017-10-10 and ICROVID: 2020-2052-BO, 2021-01-21). This subanalysis of cholinesterase activities was specified a priori in the protocols of both studies.^23,24 Patients were enrolled from May 2018 to November 2022 in the ICU of the Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital and met the Sepsis-3 criteria.²⁵ Patients were allocated to an analytical cohort depending on clinical, microbiological, and virological findings. COVID-19 and influenza were diagnosed by positive polymerase chain reaction. Written informed consent was obtained by medical doctors of the study team not involved in the patient care from either the patient or the patient's legal proxy if the patient was incapacitated. Demographic and clinical data were collected from the digital charts and files at sepsis onset (T₀), 3 ± 1 days (T₁), and 7 ± 1 days (T₂) after sepsis onset and during the entire ICU stay. Blood samples were collected at T₁ and T₂. Healthy volunteers were recruited in ICROS from May 2018 to June 2022. They were not formally matched with patients with nonviral sepsis but a target age range and gender proportion similar to that of patients with nonviral sepsis were taken into consideration during recruitment.

Definition of sepsis-associated encephalopathy

Because of the lack of concurrent diagnostic criteria^3,26 and considering definitions in other studies,^12,27–29 SAE was defined as a positive Confusion Assessment Method for the ICU (CAM-ICU) and/or Glasgow Coma Scale (GCS) < 15 and/or description of altered mental state or delirium in the doctor’s or nurse’s notes, all not attributed to other encephalopathies or medication.²⁹ Patients with other causes of encephalopathy, including structural neurologic disease, mental-state-altering medication, and metabolic disorder were excluded from analysis. Sedation was defined as the use of any dose of a sedative (e.g., midazolam, clonidine, remifentanil, sufentanil, and propofol). If the patient was under moderate or deep sedation (target Richmond Agitation Sedation Scale [RASS] < –1), these sections of the chart were not evaluated for the presence of SAE. Patients with light sedation (target RASS –1 to 0) were considered to have SAE if they showed signs of hypoactive delirium (GCS < 9) and/or hyperactive delirium.

Measurement of cholinesterase activities and laboratory parameters

Point-of-care testing of cholinesterase activity was performed with assay kits and the device LISA-ChE^® model 98LCHE003, s SN 22 (both, Dr. F. Köhler Chemie GmbH, Germany) in 10 µl ethylenediaminetetraacetic acid whole blood. The measurement is based on a colorimetric method, a variant of the Ellman method improved by Worek et al.^30,31 The concentrations of hemoglobin (Hb), albumin, and cholinesterase (ChE) were measured photometrically and C-reactive protein (CRP), procalcitonin (PCT), ferritin, and interleukin-6 (IL-6) using immunoassays at the Institute for Clinical Chemistry and Laboratory Diagnostic of Jena University Hospital.

Statistical analyses

We report median and interquartile ranges [IQRs] for continuous demographic, clinical, and laboratory data and absolute and relative frequencies for categorical variables. For intergroup comparisons, we applied Mann–Whitney U tests for continuous variables and Chi square tests for categorical variables.

Because of repeated measurements, the associations were assessed using Spearman’s rank correlation coefficients including 95% confidence intervals and P values for each study time point. We used receiver operating characteristic analysis³² to obtain thresholds (i.e., cut-offs) of AChE and BChE levels for predicting ICU mortality. The optimal cut-off was chosen based on Youden’s Index.³³

Statistical analysis was performed using Graphpad Prism 9.5.1 (GraphPad Software Inc., San Diego, CA, USA), R 4.1.0 (R Foundation for Statistical Computing, Vienna, Austria), and R-Studio 1.4.1717 (PBC, Boston, MA, USA). Missing data were few, perceived missing at random and handled with pairwise deletion. We applied a significance level of 5% and report two-sided P values.

Results

Demographic and clinical characteristics

We analyzed a total of N = 108 patients with viral sepsis from the studies ICROS (n = 51) and ICROVID (n = 57). Patients with nonviral sepsis (N = 117) and healthy volunteers (N = 81) from ICROS served as controls (Fig. 1). In patients with viral sepsis, SARS-CoV-2 (n = 106) and influenza virus (n = 2) were the cause of sepsis. In patients with nonviral sepsis, microbiological findings showed bacteria (n = 87), fungi (n = 35), and/or parasites (n = 1) as causative agents. When no pathogen was identified (n = 22), clinical and laboratory findings indicated a nonviral origin. Demographic data are summarized in Table 1. Patients with nonviral sepsis were older and had lower weight and body mass index (BMI), and higher sequential organ failure assessment scores at T₀ and T₁ than viral sepsis patients. In viral sepsis, fewer patients required vasopressors and sedation at T₁ but more patients required ventilation at T₁ and T₂ than patients with nonviral sepsis did (Table 1).

Fig. 1.

Overview of patient inclusion and analytical cohorts from the recruiting studies ICROS and ICROVID. Created with BioRender.com.

ICROS = Identification of cardiovascular and molecular prognostic factors for the medium-term and long-term outcomes of sepsis; ICROVID = Identification of cardiovascular and molecular prognostic factors for the morbidity and mortality in COVID-19-sepsis

Table 1.

Demographic and clinical data

Variable	VS N = 108	NVS N = 117	HV N = 81	P value
				VS vs NVS	VS vs HV	NVS vs HV
Age (yr), median [IQR]	61.0 [50.3–69.0]	65.0 [56.0–75.0]	65.0 [52.5–72.0]	0.01a	0.08a	0.57a
Sex (female), n/total N (%)	31/108 (29%)	41/117 (35%)	29/81 (36%)	0.38b	0.38b	0.99b
Weight (kg), median [IQR]	89.2 [80.0–110.0]	80.0 [70.0–95.5]	76.0 [65.5–87.1]	< 0.001a	< 0.001a	0.02a
BMI (kg·m−2), median [IQR]	29.7 [26.1–35.7]	28.0 [23.6–32.2]	25.3 [23.0–27.3]	0.006a	< 0.001a	0.001a
LOS-ICU (days), median [IQR]	11 [7–26]	10 [5–22]	-	0.12a	-	-
SOFA at T0 (points), median [IQR]	4 [3–7]	9 [7–11]	-	< 0.001a	-	-
SOFA at T1 (points), median [IQR]	4 [3–7]	8 [5–10]	-	< 0.001a	-	-
SOFA at T2 (points), median [IQR]	4 [2–7] (N = 100)	5 [2–8] (N = 110)	-	0.69a	-	-
Catecholamine at T1 (yes), n/total N (%)	27/108 (25%)	75/117 (64%)	-	< 0.001b	-	-
Catecholamine at T2 (yes), n/total N (%)	34/100 (34%)	38/110 (35%)	-	0.99b	-	-
Sedation T1 (yes), n/total N (%)	20/108 (19%)	55/117 (47%)	-	< 0.001b	-	-
Sedation T2 (yes), n/total N (%)	33/100 (33%)	39/110 (36%)	-	0.82b	-	-
Ventilation T1 (yes), n/total N (%)	87/108 (81%)	64/117 (55%)	-	< 0.001b	-	-
Ventilation T2 (yes), n/total N (%)	62/100 (62%)	45/110 (41%)	-	0.004b	-	-
SAE: ICU (yes), n/total N (%)	24/108 (22%)	70/117 (60%)	-	< 0.001b	-	-
Mortality: ICU (nonsurvivor), n/total N (%)	22/108 (20%)	18/117 (15%)	-	0.42b	-	-

^aMann–Whitney U test (P < 0.05 are marked in bold)

^bChi square test (with Yates’s continuity correction, P < 0.05 are marked in bold)

Catecholamine: use of any dose of dobutamine, norepinephrine, or epinephrine; Sedation: any dose of a sedative; Ventilation: any duration of invasive or noninvasive ventilation > 6 hours

BMI = body mass index; HV = healthy volunteers; ICU = intensive care unit; IQR = interquartile range; LOS-ICU = length of stay in ICU; NVS = patients with nonviral sepsis; SAE = sepsis-associated encephalopathy; SOFA = sequential organ failure assessment score; T₀ = onset of sepsis; T₁ = 3 ± 1 days after onset of sepsis; T₂ = 7 ± 1 days after onset of sepsis; VS = patients with viral sepsis

There were no differences between patients with viral sepsis and nonviral sepsis in comparison with healthy volunteers regarding age and sex (Table 1). Patients with viral sepsis and nonviral sepsis had a higher weight and BMI than healthy volunteers.

Acetylcholinesterase and butyrylcholinesterase activities during the acute phase of sepsis

We measured the activities of AChE and BChE in whole blood of patients with sepsis and healthy volunteers and report AChE activity per hemoglobin as well as hemoglobin concentrations (Fig. 2). Patients with viral sepsis and nonviral sepsis showed lower activities in AChE and BChE than healthy volunteers at both study time points (Fig. 2A and 2B). Patients with nonviral sepsis showed lower levels of both enzyme activities than patients with viral sepsis at both time points (Fig. 2A and 2B). Acetylcholinesterase activity per hemoglobin was higher in patients with nonviral sepsis than in healthy volunteers at T₁ (Fig. 2C). Hemoglobin was reduced in patients with viral sepsis and nonviral sepsis at both time points compared with healthy volunteers, with lower concentrations in nonviral sepsis compared with viral sepsis (Fig. 2D).

Fig. 2.

Comparison of patients with viral sepsis, patients with nonviral sepsis at T₁ (3 ± 1 days after the onset of sepsis) and T₂ (7 ± 1 days after the onset of sepsis), and of healthy volunteers. (A) AChE activity, (B) BChE activity, (C) AChE activity per hemoglobin, and (D) hemoglobin. Data are displayed as scatter dot plots with median and interquartile range.

*P < 0.05 (Mann–Whitney U test)

AChE = acetylcholinesterase; BChE = butyrylcholinesterase

Acetylcholinesterase and butyrylcholinesterase activities in patients with versus without sepsis-associated encephalopathy

Twenty-four patients with viral sepsis (22%) and 70 patients with nonviral sepsis (60%) developed SAE during their stay in the ICU; there was no significant difference in the ICU length of stay (Table 1). We compared the activity levels of AChE and BChE at T₁ and T₂ in patients with and without SAE (Fig. 3). Patients with viral sepsis who developed SAE had decreased AChE activity at T₁ (Fig. 3A). Patients with nonviral sepsis who developed SAE in the ICU had lower BChE activity at T₁ (Fig. 3B).

Fig. 3.

Comparison of patients with viral sepsis and nonviral sepsis with versus without sepsis-associated encephalopathy during their stay in the intensive care unit at T₁ (3 ± 1 days after the onset of sepsis) and T₂ (7 ± 1 days after the onset of sepsis). (A) AChE activity, (B) BChE activity. Data are displayed as scatter dot plots with median and interquartile range.

*P < 0.05 (Mann–Whitney U test)

AChE = acetylcholinesterase; BChE = butyrylcholinesterase; ICU = intensive care unit; SAE = sepsis-associated encephalopathy

Acetylcholinesterase and butyrylcholinesterase activities in intensive care unit survivors versus nonsurvivors

Twenty-two patients with viral sepsis (20%) and 18 patients with nonviral sepsis (15%) died in the ICU (Table 1). We compared the activity levels of AChE and BChE at T₁ and T₂ in patients who survived the ICU and those who did not (Fig. 4). Acetylcholinesterase showed no differences (Fig. 4A). In both viral sepsis and nonviral sepsis, nonsurvivors had decreased activity of BChE at T₂ compared with survivors (Fig. 4B). In nonviral sepsis, reduced activity of BChE was also observed at T₁. Nonetheless, the diagnostic accuracy of the obtained cut-offs was in the moderate range (Electronic Supplementary Material [ESM] eTables 1 and 2).

Fig. 4.

Comparison of ICU survivors and nonsurvivors of patients with viral sepsis and nonviral sepsis at T₁ (3 ± 1 days after the onset of sepsis) and T₂ (7 ± 1 days after the onset of sepsis). (A) AChE activity, (B) BChE activity. Data are displayed as scatter dot plots with median and interquartile range.

*P < 0.05 (Mann–Whitney U test)

AChE = acetylcholinesterase; BChE = butyrylcholinesterase; ICU = intensive care unit

Laboratory parameters and their association with cholinesterase activities

We measured the concentrations of parameters previously reported to affect cholinesterase activity (Table 2). These parameters include Hb because of the presence of AChE on erythrocytes,³¹ the concentration of plasma cholinesterase (ChE, common designation of BChE), and albumin as a surrogate parameter of synthetic liver function.³⁴ Patients with nonviral sepsis showed lower levels of Hb, ChE, and albumin than patients with viral sepsis (Table 2) at both time points. We assessed the association of these parameters with the cholinesterase activities in viral (Table 3) and nonviral (ESM eTable 3) sepsis. In patients with viral sepsis, there was a weak positive association between AChE activity and Hb, between AChE activity and albumin, and between AChE activity and ChE, while BChE activity was positively associated with albumin and with ChE (Table 3).

Table 2.

Descriptive statistics of laboratory data of patients with viral sepsis, patients with nonviral sepsis (T₁ and T₂), and healthy volunteers

Variable	Time	VS	n	NVS	n	HV	n	P valuea
								VS vs NVS	VS vs HV	NVS vs HV
AChE (U·L−1), median [IQR]	T1	5,105 [4,010–6,250]	108	4,424 [3,630–5,055]	117	6,693 [5,401–8,020]	81	< 0.001	< 0.001	< 0.001
	T2	4,766 [3,732–6,235]	99	4,189 [3,397–4,783]	108	-	-	< 0.001	< 0.001b	< 0.001b
BChE (U·L−1), median [IQR]	T1	1,943 [1,393–2,468]	108	1,095 [834–1,526]	117	2,645 [2,198–3,478]	81	< 0.001	< 0.001	< 0.001
	T2	1,932 [1,427–2,595]	99	1,114 [864–1,549]	108	-	-	< 0.001	< 0.001b	< 0.001b
Hb (mmol·L−1), median [IQR]	T1	7.4 [6.4–8.0]	108	5.3 [4.9–5.8]	116	9.1 [8.6–9.6]	81	< 0.001	< 0.001	< 0.001
	T2	6.9 [5.8–7.7]	98	5.3 [4.9–5.9]	103	-	-	< 0.001	< 0.001b	< 0.001b
Albumin (g·L−1), median [IQR]	T1	30.0 [25.0–32.0]	107	21.0 [17.5–25.0]	117	43.0 [40.0–46.0]	81	< 0.001	< 0.001	< 0.001
	T2	29.0 [25.0–33.0]	94	21.0 [17.0–26.3]	106	-	-	< 0.001	< 0.001b	< 0.001b
ChE (µmol·L−1), median [IQR]	T1	88.0 [71.0–105.0]	107	39.0 [26.0–57.0]	117	146.0 [129.5–165.0]	81	< 0.001	< 0.001	< 0.001
	T2	87.0 [64.0–109.3]	94	42.0 [30.0–58.0]	103	-	-	< 0.001	< 0.001b	< 0.001b
CRP (mg·L−1), median [IQR]	T1	63.4 [32.5–124.4]	108	200.8 [129.9–285.7]	116	2.0 [0.6–2.2]	81	< 0.001	< 0.001	< 0.001
	T2	34.6 [14.7–80.6]	98	102.2 [63.8–157.3]	101	-	-	< 0.001	< 0.001b	< 0.001b
PCT (ng·L−1), median [IQR]	T1	0.18 [0.09–0.48]	108	3.80 [1.07–13.92]	116	-	-	< 0.001	-	-
	T2	0.14 [0.07–0.32]	98	0.89 [0.33–2.74]	106	-	-	< 0.001	-	-
Ferritin (µg·mL−1), median [IQR]	T1	1,287.2 [833.6–2,151.6]	104	-	-	-	-	-	-	-
	T2	1,310.3 [696.3–1,816.2]	94	-	-	-	-	-	-	-
IL-6 (pg·mL−1), median [IQR]	T1	31.4 [10.3–82.4]	105	-	-	-	-	-	-	-
	T2	16.5 [6.4–160.0]	95	-	-	-	-	-	-	-

^aMann–Whitney U test (P < 0.05 are marked in bold)

^bData of patients with viral or nonviral sepsis from T₂ was compared with healthy controls (T₁)

AChE = acetylcholinesterase; BChE = butyrylcholinesterase; CRP = C-reactive protein; ChE = cholinesterase; Hb = hemoglobin; HV = healthy volunteers; IL-6 = interleukin-6; IQR = interquartile range; NVS = patients with nonviral sepsis; PCT = procalcitonin; T₁ = 3 ± 1 days after onset of sepsis; T₂ = 7 ± 1 days after onset of sepsis; VS = patients with viral sepsis

Table 3.

Associations between AChE and BChE activities with inflammatory markers and laboratory data of patients with viral sepsis

Variable	AChE						BChE
	Time	ρa	95% CI		P value	n	ρa	95% CI		P value	n
Hb (mmol·L−1)	T1	0.51	0.35	0.63	< 0.001	108	0.13	−0.06	0.31	0.19	108
	T2	0.38	0.19	0.54	< 0.001	97	0.20	0.00	0.38	0.049	97
Albumin (g·L−1)	T1	0.23	0.04	0.40	0.02	107	0.36	0.19	0.52	< 0.001	107
	T2	0.29	0.09	0.47	0.004	93	0.35	0.16	0.52	< 0.001	93
ChE (µmol·L−1)	T1	0.19	0.00	0.37	0.048	107	0.55	0.40	0.67	< 0.001	107
	T2	0.23	0.03	0.41	0.03	93	0.51	0.34	0.65	< 0.001	93
PCT (ng·L−1)	T1	−0.17	−0.35	0.02	0.08	108	−0.29	−0.45	−0.11	0.002	108
	T2	−0.17	−0.36	0.03	0.09	97	−0.27	−0.44	−0.07	0.008	97
CRP (mg·L−1)	T1	−0.19	−0.36	0.00	0.05	108	−0.25	−0.42	−0.06	0.01	108
	T2	−0.24	−0.42	−0.05	0.02	97	−0.34	−0.51	−0.15	< 0.001	97
Ferritin (µg·mL−1)	T1	0.16	−0.03	0.35	0.10	104	0.05	−0.15	0.24	0.62	104
	T2	0.05	−0.15	0.25	0.63	93	−0.01	−0.21	0.20	0.95	93
IL–6 (pg·mL−1)	T1	−0.03	−0.22	0.16	0.75	105	−0.06	−0.25	0.14	0.57	105
	T2	−0.21	−0.40	−0.01	0.04	94	−0.26	−0.44	−0.06	0.01	94

^aSpearman’s rank correlation coefficient with 95% CI and P value (P < 0.05 are printed in bold)

AChE = acetylcholinesterase; BChE = butyrylcholinesterase; CI = confidence interval; CRP = C-reactive protein; ChE = cholinesterase; Hb = hemoglobin; IL-6 = interleukin-6; PCT = procalcitonin; SOFA = sequential organ failure assessment score; T₁ = 3 ± 1 days after onset of sepsis; T₂ = 7 ± 1 days after onset of sepsis

We further measured the concentration of inflammatory markers—CRP, PCT, ferritin, and IL-6—in patients with viral sepsis, and compared the concentrations of CRP and PCT with those in patients with nonviral sepsis. We also performed association analyses with cholinesterase activities (Table 2 and Table 3). Patients with viral sepsis showed lower levels of PCT and CRP than patients with nonviral sepsis. In viral sepsis, BChE activity showed more negative associations with inflammatory markers than AChE activity did (Table 3).

Discussion

Cholinergic imbalance in viral and nonviral sepsis

A modulation of the cholinergic anti-inflammatory pathway has been proposed as a mechanism for developing organ dysfunction in severe COVID-19.³⁵ Nevertheless, studies in patients with viral sepsis²² are scarce. In patients with viral and nonviral sepsis, we found a reduction in both AChE and BChE activity compared with healthy volunteers. These reductions were more pronounced in patients with nonviral sepsis. To our knowledge, this is the first description of a reduction in AChE activity in viral sepsis and the first direct comparison with nonviral sepsis, providing new evidence for a possible cholinergic imbalance independent of causative pathogens. Our results are in line those of a study finding reduced levels of BChE activity in 54 patients with severe COVID-19 compared with 40 historical healthy controls.²² Furthermore, our results are consistent with those of other studies in critically ill patients³⁶ and patients with sepsis of any origin³⁷ that found a reduction of BChE activity within the first six³⁷ or 28³⁶ days after the onset of severe inflammation and sepsis.

Acetylcholinesterase and butyrylcholinesterase activities in sepsis-associated encephalopathy

We found that patients with viral sepsis who developed SAE in the ICU had lower AChE activity on day 3 after the onset of sepsis compared with patients without SAE. This finding was not observed in patients with nonviral sepsis who developed SAE in the ICU. These results are in line with those of a previous study,¹² showing a significant decrease of AChE activity after three days in patients with sepsis of any origin and suspected SAE. Nevertheless, the authors did not compare AChE activities between patients with and without SAE. Our findings indicate that the significance of AChE activity as a prognostic marker during acute sepsis may be more relevant to viral sepsis than to nonviral sepsis.

In patients with nonviral sepsis with SAE we observed decreased BChE activity on day 3 of sepsis compared with patients without SAE—consistent with other reports of lower levels of BChE associated with postoperative delirium in patients undergoing hip replacement³⁸ and with critical illness.³⁹ Our results indicate that AChE activity in the early phase of sepsis may be of prognostic value for the development of SAE in viral sepsis and BChE activity on day 3 in nonviral sepsis. These findings should be explored further and validated in future studies.

Acetylcholinesterase and butyrylcholinesterase activities and intensive care unit mortality

Acetylcholinesterase activities showed no differences between survivors and nonsurvivors in viral sepsis. Hence, AChE appears unsuitable for predicting mortality, consistent with our results in nonviral sepsis and the literature.⁴⁰ Butyrylcholinesterase activity was lower seven days after the onset of sepsis in ICU nonsurvivors in both viral sepsis and nonviral sepsis. The plasma concentration⁴¹ and the activity²² of BChE have been shown to correlate with disease severity and mortality in severe COVID-19, which we have reproduced in a larger cohort. This supports BChE as a prognostic marker in patients with sepsis. Zivkovic et al. found values of < 1,661 U·L⁻¹ measured at sepsis onset using a similar device to predict mortality (sensitivity 94%; specificity 48%) in patients with sepsis of any origin.³⁷ We found cut-off values with lesser accuracy (ESM eTables 1 and 2) potentially explained by methodological differences in the device used and the timing of measurements. This illustrates both the need for specific cut-off values depending on the diagnostic device and the time dependency of measurements.

The association of butyrylcholinesterase activity and systemic inflammation

A hypothesized pathophysiologic link between increased mortality, organ dysfunction, and reduced BChE activity is inflammation.^18,36,42 This link may also hold true for patients with viral sepsis, as BChE activity was negatively correlated with PCT, CRP, and IL-6 in our exploratory study. Our results are consistent with findings in critically ill patients³⁶ showing a negative association with reduced activity of BChE and inflammatory markers. As the association between BChE activity in blood and systemic inflammation does not prove inflammation and cholinergic imbalance in the brain, further possibly preclinical studies are needed to investigate this pathophysiologic link.

Further associations with acetylcholinesterase and butyrylcholinesterase activities

In our study, Hb was reduced in all patients with sepsis. Patients with nonviral sepsis had lower Hb than patients with viral sepsis. We found a weak association between AChE activity and Hb, potentially explained by the presence of AChE in erythrocytes,³¹ implying Hb as a confounding factor. The employed measurement method reduced the influence of Hb on obtained AChE activities by adapting the measurement wavelength.³¹ Erythrocytic AChE only contributes to part of the total AChE activity in blood (up to 44%); the other part is derived from nonerythrocytic sources.¹⁴ Therefore, the influence of Hb on AChE activity may be limited. Previous studies have assessed the influence of Hb on delirium in critical illness. Anemia presented a risk factor for increased 28-day mortality but was not associated with neurologic dysfunction in a large multicentre cohort study.^43,44 We showed positive associations between BChE activity, its concentration in plasma (ChE), and albumin. Butyrylcholinesterase is produced by the liver,⁴⁵ so its activity is decreased in liver disease, trauma, and sepsis.^18,34,37,46 Liver disease has been identified as a risk factor for delirium independent of hepatic encephalopathy⁴⁷ and a higher prevalence of liver failure has been shown in patients developing SAE.²⁸ Our results indicate a potential link between liver failure and SAE through cholinergic imbalance, which should be examined further in future studies.

Limitations and strengths of the study

The obtained results should be evaluated in light of the limitations of this study. Recruitment was from one study centre and the study size was limited. The applicability of results to other regions could be limited. The exclusion criteria of the recruiting studies^23,24 yielded a selected cohort of patients, which may limit the generalizability of our results in patients with severe heart disease. The incidence of COVID-19 during recruitment resulted in a high prevalence thereof in the viral sepsis cohort, potentially limiting the applicability of results to other viral infections. Further, Hb and hepatic function may present confounding factors: the prognostic value may be different in patients with anemia and acute or chronic liver disease. The control group of patients with nonviral sepsis showed different patient characteristics; thus, the results should be confirmed in matched cohorts. Different characteristics may result from pathophysiologic differences and predisposing factors in viral and nonviral sepsis. Moreover, the impact of medication such as vasopressors on cholinesterase activity was not assessed. The inability to assess SAE in patients with moderate to deep sedation limits the generalization of findings for sedated patients. Despite its limitations, this study represents the hitherto largest cohort, in which cholinergic imbalance was investigated in viral sepsis. Furthermore, the recruitment of all patients was prospective and the analysis prespecified in published protocols. The analysis not only encompasses comparisons of cohorts but also outcome-relevant subanalyses which may increase the clinical relevance of the results.

Conclusion

The enzymatic activities of AChE and BChE in patients with viral and nonviral sepsis were reduced during the acute phase of sepsis. Patients with viral sepsis who developed SAE in the ICU expressed a reduced activity of AChE. Furthermore, ICU nonsurvivors of both cohorts showed a reduced BChE activity seven days after onset of sepsis, indicating a potential prognostic relevance of BChE.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

Open Access funding enabled and organized by Projekt DEAL.