Septic shock is a leading cause of mortality in criticallyill patients despite the use of new antibiotics and resuscitation therapies [1, 2]. Given the significance of timely beginning of appropriate antibiotics to optimize septic shock outcomes [1, 2], clinicians must quickly achieve a diagnosis of infection. In fact, it is now accepted that starting an early effective antibiotic therapy in the course of an infection decreases morbidity and mortality in this specific condition [2, 3]. Moreover, more than diagnosing sepsis, clinicians are also faced with the mission of monitoring the patients’ responses to therapy toward either the resolution of the illness or its progression into a multisystem organ failure and finally death.
For all of these reasons, there is a need to develop biomarkers of sepsis in order to increase clinical assessments, differentiate between bacterial and non-bacterial infection, and to differentiate those patients who are most likely going to recover from those who face a rather bad outcome. So far, many bio-markers have the potential to serve a decisive role by providing adjunctive information to guide clinicians to a rapid diagnosis and to adjust a treatment. There are hundreds of biomarkers which could be potentially used for diagnosis and prognosis in septic patients . Moreover, and to the best our knowledge, Procalcitonin (PCT) and C-reactive protein (CRP) have been the most widely investigated [1, 2].
More recently, it was established that The Serum cholinesterase (SchE) activity level was significantly low in patients with septic shock due to bacterial infections [2, 4, 5]. Moreover, in the ICU, a serial measurement of sepsis biomarkers has been used as an adjunctive tool and as a guide to discontinue the antimicrobial therapies earlier [6-8]. However, up to date, a few studies have evaluated the discriminative power of PCT, CRP, and Serum cholinesterase (SchE) kinetics as predictors of survival outcome in patients admitted to the ICU with septic shock [2, 4, 5, 8- 12]. In this study, we aim to explore the value of PCT, CRP, andserum SchE activity kineticsas useful predictors of mortality in patients with septic shock admitted in ICU.
There is no conflict of interest related to the research described above. This study was approved by the institutional review board, and the patients, or their relatives, gave written informed consent after being fully informed about the protocol. We conducted a prospective single-blinded study in an intensive care unit (ICU) of a university hospital during a period of one year. Were included all patients with 18 years of age, or older, with septic shock. Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection.
Septic shock was defined according to the Third International Consensus Definitions for Sepsis and Septic Shock . The patient was considered to be in shock if the mean arterial pressure was less than 65 mmHg or the systolic blood pressure was less than 90 mmHg despite an adequate infusion of fluids (at least 30 ml/kg of crystalloids or 500 ml of colloids) in presence of tissue hypoperfusion signs . Patients with septic shock were clinically defined as those needing a vasopressor administration to maintain a mean arterial pressure of 65 mm Hg, or greater, and serum lactate level greater than 2 mmol/L (>18 mg/dl) in the absence of hypovolemia. All included patients were monitored with an arterial catheter and explored by an echocardiography (by the same physician).
A documented infection was diagnosed when there was a clinically evident source of infection. The diagnosis of bacterial infection was confirmed when a microorganism(bacterium) was isolated in various bacterial samples. In our study, microbiological analyses were performed on blood samples, urine and sputum cultures, and specimens taken from other sites according to the clinical suspicion regarding the source of sepsis. All the patients underwent chest radiography and/or CT scanning as requested. Were excluded all the patients with any of the following conditions: previous diagnosis of chronic and acute liver disease, cirrhosis, active tuberculosis, cancer, malnutrition; organophosphate poisoning and patients with brain herniation and/or declared brain-dead. Moreover, were excluded patients with cardiogenic shock and those with hypovolemic shock (hemorrhagic shock). Finally, we excluded all the patients with mixed shock (association cardiogenic and septic shock in the same patient). Were also excluded, all the patients with viral and/or fungal infection.
The clinical data were prospectively collected for each patient using pre-printed case report forms. For all included patients, a data entry form was designed to collect demographic, clinical, biological and radiological data on admission and during ICU stay. The systemic inflammatory response syndrome (SIRS) [9, 13] was checked on admission and during ICU stay.The biochemical parameters measured on admission and during the ICU stay were arterial blood gases and acid–base status (pH and bicarbonate), Blood urea nitrogen (BUN), creatinine blood, leukocytes counts, haemoglobin concentration and coagulation profile.
The severity of illness was assessed by simplified acute physiology score (SAPS II) calculated within 24 h of admission , and according to the SOFA score calculated on ICU admission and during ICU hospitalization . During the ICU stay, all complications were recorded: nosocomial infections , thrombocytopenia, gastrointestinal bleeding, arrhythmias and metabolic complications. Patients were followed until ICU discharge or death. All vital signs, standard laboratory variables, results for cultures of specimens from new infection sites, dose of vasopressors, and interventions were recorded daily.
Biomarkers: for all included patients, blood samples of septic biomarkers (PCT, SchE activity, and C-reactive protein (CRP)) were obtained. Serum was collected for CRP, SchE activity and PCT assays upon ICU admission (day 0: D0), at the day of septic shock (day 1: D1), then 3 and 5days after the septic shock development (D3 and D5 respectively).All the members of our disciplinary team were blinded to the SchE activity, procalcitonin and CRP values. All the biomarkers’ assays were processed at the same laboratory.
Data were entered centrally by a healthcare worker using the SPSS version 18.0 for Windows. Categorical data were expressed in proportion and subgroups, and continuous variables were expressed as means (± SD). Subgroups (survivals and deaths) were analyzed by the Chi-square test (or Fisher\’s exact test), the two-group t-test, or the Mann–Whitney-U test, as appropriate. The level of significance was set at p < 0.05. Sensitivity, specificity, and predictive values of SchE activity, PCT and CRP for discrimination between survivals and deathswere calculated. Moreover, Kinetics of SchE activity, PCT and CRP were defined by the variation of values between the day of septic shock development (D1), D3andD5. They were calculated by the ratio of the difference between the second and the first measurement [e.g. (value on D3 – value on D1)]. These variables were then defined as Δ-CRPD1-D3, and Δ-CRPD1-D5, respectively. The best cut-off values for serum SchE activity, PCT, CRP, Δ-CRP, Δ-PCT, and Δ- serum-ChE were chosen as the values that optimized sensitivity and specificity. Receiver operating characteristic (ROC) curves were plotted, and the respective areas under curves were calculated. In a ROC curve, the true-positive rate (sensitivity) is plotted against the false-positive rate (1-specificity), with the area under the curve being proportional to the probability of a correct discrimination. Risk factors are evaluated in univariate analysis and by multivariate analysis by a multiple logistic stepwise regression procedure. Odds ratios are estimated from the b coefficients obtained, with respective 95% confidence intervals (CI 95%).
Descriptive characteristics and outcome of Patients During the study period, 60 patients were included and 92 patients were excluded (Figure 1). The mean age was 47.7 ± 19 years. There were 46 male (74%) and 14 female (26%) patients. Mean SAPSII on ICU admission was 40.7 ± 16 (median: 37) and mean SOFA score on ICU admission was 16 ± 4 (median: 7). Moreover, 44 patients (73%) had one or more co-morbid-conditions. The most common past medical diseases were: arterial hypertension in 20 patients (33%), diabetes mellitus in 18 (30%), chronic heart disease in 17 (28.3%) and chronic obstructive pulmonary disease (COPD) in 9(15%).
On ICU admission, 36 patients (60%) developed circulatory failure needing a vasopressor administration (norepinephrine) to maintain a mean arterial pressure of 65 mm Hg or greater. Moreover, 57 patients (95%) had a respiratory failure requiring invasive mechanical ventilation on ICU admission. Finally, 26 patients (43.3%) developed acute renal failure on ICU admission. The clinical presentations of the study group on admission are shown in Table 1.
During their ICU stay, all patients developed septic shock due to a bacterial infection. The most common sites of infection were the lungs in 36 patients (60%), followed by abdominal infections in 6(10%), wound infections in 5(8.3%), urinary tract infections in 4 (6.6%), bloodstream infections in 4 (6.6%) and other localizations in 5 cases (8.3%).The most identified pathogens were: Klebsiella pneumoniae in 12 patients (20%), Acinetobacter baumannii in 11 (18.3%), Pseudomonas aeruginosa in 6 (10%) and Escherichia coli in 4 (6.6%). Within the study period, out of the 60 included patients, 37 patients died (61%). The comparison between the two groups (deaths and survivors) showed that the factors associated with poor outcome were age, SOFA score on ICU admission and the need of invasive mechanical ventilation (Table 2).
Value of Sepsis-biomarkers and their kinetics to predict the outcome
The analysis of sepsis-biomarkers plasma concentrations showed that there was no difference in the mean of plasma SchE activity, nor in PCT and CRP plasma concentrations between survivors and non-survivors on ICU admission(D0) and the day of septic shock(D1)(Table 3).Moreover, as shown in Table 3, the comparison of mean plasma SchE activity, PCT and CRP plasma concentrations (on D3 and D5) between survivors and non-survivors, showed a significant difference between the two groups (deaths and survivors). On ICU admission (D0) and on the day of septic shock (D1), there was no difference in the mean of PCT and CRP plasma concentrations between survivors and non-survivors(Table 3).
A significant decrease of mean PCT and CRP values between admission, D3 and D5 was found in survivors, compared with non-survivors, with a significant difference (P<0.05) (Figures 2 and 3).Furthermore, a significant increase of mean SchE activity value between admission, D3 and D5 was found in survivors, compared with non-survivors, with a significant difference (P3270 UI/L on D5 was associated with good outcome with sensitivity at 71%, specificity at 91%, and area under the curve: 0.83. A ROC analysis identified that a decrease of Δ-PCT (D1-D5) by more than 7.8 mg/dl (area under the curve: 0.65; S: 35%and Sp: 85%) and a decrease of Δ-CRP (D1-D5) by more than 49 mg/l (area under the curve: 0.62; S: 57% and sp: 75%) as the most accurate cut-offs in predicting a good outcome.
On the other hand, we found that a decrease of Δ- SchE activity (D1-D5) by more than 820 UI/l was associated with a bad outcome with a sensitivity at 71%, a specificity at 81% and an area under the curve at 0.83 (figure 5).Moreover, a significant correlation was found between Δ-PCT (D1-D3) and Δ- SchE activity (D1-D3) (p: 0.001, R: – 0.44); and Δ-PCT (D1-D5) and Δ- SchE activity (D1-D5)(p: 0.026, R: – 0.33)(Figure 6).The multivariate analysis showed that the factors associated with the poor outcome (death) were age of more than 63 years (p=0.04; OR: 1.08; 95%CI: 1.004-1.16) and a Δ- SchE activity (D1-D5) of more than 820 UI/l (p=0.01; OR: 1.003; 95%CI: 1.0 -1.005).
Severe sepsis and septic shock are among the leading causes of morbidity and mortality among critically ill patients, thus, the detection of prognostic factors is fundamental to determine their outcome. Almost all included patients in our study showed elevated CRP, PCT and low cholinesterase activity (SchE) levels on ICU admission. However, the initial measurements of these inflammatory biomarkers failed to distinguish between the survivors and the non-survivors. On the other hand, our study confirms that kinetics of PCT, CRP, and cholinesterase activity (SchE) can be used as useful predictors of mortality in patients with septic shock admitted in ICU. As a consequence, our study results highlight the validity and prognostic value of the kinetics of sepsis biomarkers (PCT, CRP, andcholinesterase (SchE) activity in our study) in the detection of high risk, life-threatening complications in patients who suffer from septic shock due to a bacterial infection.
Sepsis is a heterogeneous syndrome characterized by a complex immune-inflammatory response to presumed or proven infection . Early identification and intervention are crucial to improve sepsis outcomes. Leaving clinical diagnosis away, in many situations the laboratory diagnostics (sepsis biomarkers) may represent a help tool to predict the positive diagnosis of sepsis and/or septic shock [2,3, 6]. There are hundreds of biomarkers which could be potentially used for diagnosis and prognosis in septic patients .However, few studies have evaluated the discriminative power of PCT, CRP, and Serum cholinesterase (SchE) kinetics as predictors of survival outcome in patients admitted to the ICU with septic shock [2-5, 10-12].
The prognostic value of CRP and PCT kinetics has been studied in several types of infection, with mortality as the main outcome variable with contradictory results [8-11, 18, 19].In our study, we found a significant decrease of mean PCT and CRP values on D3and D5 in comparison with D1 (day of the septic shock) in survivors compared with non-survivors. In fact, when a patient improves under symptomatic treatment and antibiotics, the inflammatory reaction decreases and the value of these sepsis-biomarkers should decline. More recently, it was established that The Serum cholinesterase (SchE) activity level had significantly decreased in patients with septic shock due to bacterial infections [2, 4, 5, 12, 20]. Moreover, the reduction in the SchE activity was detected significantly earlier compared to those of routinely measured inflammatory biomarkers . In fact, the reduction in SchE activity was observed as early as 1-2 hours following the inflammatory and/or sepsis onset .
The pathophysiology of the rapid decline of SchE activity in patients with septic shock is not well-understood and several hypotheses were postulated . In all cases, the role of inflammatory reaction is well-established [2, 4, 5, 12]. The first hypothesis is during the progression of the sepsis, bacteria and their toxins could activate neutrophils to release various substances (such as proteases, cytokines …) affecting liver function with reduced synthesis of SchE. The second is the dilution effect due to fluid therapy leading to a decrease in the plasmatic concentration of SchE. The third is the increased catabolism of SchE and the inhibition of SchE by inflammatory mediators (cytokines). The last is the increased transcapillary loss of SchE due to the increase of capillary permeability caused by a sepsis.
In one recent prospective study, Bahloul et al  showed that decreased SchE activity can be used as a reliable diagnostic marker for septic shock. More recently, it has been established that the Serum cholinesterase (SchE) activity level can be used as prognostic biomarker in septic shock [10, 4, 5]. In one prospective observational study , Zivkovic AR et al  showed that the SchE enzyme activity was markedly reduced in all septic patients during the observation period, and the measured SchE enzyme activity in non-survivors was notably lower than that of the surviving patients for the duration of 28 days.
Our study confirms the results of these studies [4, 5, 12], in fact, we found a significant increase of mean SchE activityvalues between admission, D3 and D5 in survivors, compared with non-survivors. Moreover, we found that a decrease of Δ- SchE activity (D1-D5) by more than 820 UI/l was associated with a bad outcome with a sensitivity at 71%, a specificity at 81% and an area under the curve at 0.83. When a patient improves under symptomatic treatment and antibiotics, the inflammatory reaction decreases, and SchE activity increases with time [2, 4, 5, 12]. However, in severe cases with multi-organ failure with worse outcome, the inflammatory reaction will be more pronounced, explaining the progressive decrease of SchE activity.
Based on our study and some other data detailed above, we recommend that an estimation of SchE activity, procalcitonin and CRP the day of septic shock, followed by estimation within the next 72–120 h could help the prognostic assessment of critically ill patients with severe sepsis/septic shock. The small size of our sample and the high rate of excluded patients can represent a methodological limitation. However, our study represents the second large study exploring prognostic value of serum cholinesterase activity in the septic shock after the study published by Peng ZL et al . Moreover, many patients died before the second sample could be taken; hence, our hypothesis could not be tested in all the patients.
Our study suggests that, in a group of critically ill patients with severe septic shock, a rise, or no change, in procalcitonin and/or CRP level; and/or a decrease or no change in SchE activity should warn the clinician about the insufficiency and/or inadequacy of the therapy. However, a fall in procalcitonin and/or CRP levels; and/or a rise in SchE activity were associated with a favorable prognosis. Further studies are needed on this subject.