Plasma-Calprotectin as an Indicator for Immediate Need of Intensive Care Treatment in Suspected Sepsis

Introduction: Decisions regarding need of transfer to intensive care of patients with sepsis in the emergency department is challenging. We hypothesised that the new biomarker plasma-calprotectin could be used to help select patients who need intensive care, since it already has shown to be a promising tool in the intensive care unit. Methods: This prospective study was performed on consecutive sepsis alert patients. The alert summons a multidisciplinary team of physicians from the emergency department, the Department of Infectious Diseases, and the intensive care unit, who evaluate patients for possible infection and decide where to transfer the patient. Blood sampling was performed on consecutive sepsis alert patients. C-reactive protein, procalcitonin, neutrophils, and lymphocytes were routinely analysed, p-calprotectin was analysed from frozen plasma samples using a specic turbidimetric assay. Results: Among 367 sepsis alert patients, 335 had an infection of whom 66 were immediately transferred to the intensive care unit or high dependency unit. 269 patients were transferred to ordinary wards. Median p-calprotectin for all infected patients was 2.2 (IQR 1.2–3.9), 3.3 (IQR 1.6–5.2) among those transferred immediately to intensive care unit/ high dependency unit and 2.1 (IQR 1.1–3.5) among those transferred to wards (p = 0.0001). Analysis of area under the receiver-operating characteristic (ROC) curve for transferral to higher care level showed superiority for p-calprotectin compared to procalcitonin and neutrophil-lymphocyte-ratio, both regarding all sepsis alert cases and regarding the patients with infection (p < 0.001 for all comparisons)). The best p-calprotectin cut-off 4.0 mg/L showed sensitivity 42.5% and specicity 83% for transferral to ICU/HDU among patients with infection. Conclusion: In sepsis alert patients, p-calprotectin was signicantly elevated in patients transferred intensive was superior to traditional as a predictor of need for intensive care.


Background
Sepsis is a frequent reason for hospital care and a leading cause of death, with a mortality ranging from 12-20%, in the western world (1)(2)(3)(4). According to a study from Rudd et al, sepsis counted for 20% of all global deaths in 2017 (5). The World Health Organisation has therefore highlighted early identi cation of sepsis as a global key challenge in order to provide for better treatment and improved survival rates (6).
Early recognition and treatment of sepsis have been shown to improve outcome for patients but is still a challenge for most hospitals (7,8). There is a need for better biomarkers aimed to support clinical decision making at all levels of sepsis care.

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The Surviving Sepsis Campaign include lactate and procalcitonin (PCT) as the only recommended biomarkers for sepsis care.
Lactate has been found to be a useful screening tool for severe infection and procalcitonin can support discontinuation of empirical antibiotic therapy (9). PCT as well as C-reactive protein (CRP) have diagnostic properties and are tools to distinguish infectious from non-infectious conditions. The prognostic properties of these two biomarkers have been extensively evaluated, but no clear association between them and disease severity (or outcome) has been established (10).
In the early course of sepsis, neutrophils are often activated and elevated and lymphocytes are often decreased (11). Thus, neutrophil-lymphocyte ratio (NLR) has been identi ed as a useful diagnostic marker of sepsis and has also been shown to have prognostic properties (12,13).
Calprotectin is a protein presented in the cytoplasm of neutrophils and is also expressed on the membrane of monocytes. During activation of neutrophil granulocytes, calprotectin is released into the circulation. Calprotectin has been found to increase in the bloodstream within hours in response to bacteria or endotoxin (14). Thus, calprotectin in blood samples (plasma or serum) has been proposed as a potential biomarker of sepsis (15)(16)(17)(18)(19)(20). Recently Bartakova et al found similar performance of PCT, CRP, and NLR for detection of bacterial sepsis in a hospitalized cohort, whereas calprotectin was found to have superior performance (18). Among patients treated in intensive care unit (ICU), another study found calprotectin to be superior to PCT for detection of sepsis and for discriminating sepsis from non-septic conditions. With a cut-off of 1.1 mg/L, calprotectin showed sensitivity 80% and speci city 46% for 30-day mortality (20). Calprotectin has also in a very resent study shown to be a promising biomarker for discrimination sever covid-19 from mild (21). These trials motivate further studies of the prognostic usefulness of calprotectin.
Thus, in the present study, we aimed to focus on patients with suspected sepsis in the emergency department (ED) and evaluate if p-calprotectin was superior to PCT, CRP, and NLR in predicting need of direct transferral to ICU or high dependency unit (HDU), as decided by a multidisciplinary team.

Study design
The study was performed at Karolinska University Hospital Huddinge, Stockholm, Sweden, a tertiary care hospital with approximately 700 beds. In the hospital´s ED, which has 75,000 visits per year, all patients are routinely subjected to triage with the Rapid Emergency Triage and Treatment System (22). Additionally, since September 2017, a sepsis alert has been implemented, triggered when patients show signs of organ dysfunction combined with symptoms of infection, i.e. fever, history of fever, or clinical suspicion of infection, as we have previously described (23).
Signs of organ dysfunction are either one of A or B: 1. A) at least one of: oxygen saturation below 90% despite supplemental oxygen administration, respiratory rate greater than 30 per minute, heart rate greater than 130 beats per minute, systolic blood pressure under 90 mmHg, or Glasgow Coma Scale below 8; or 2. B) blood lactate greater than 3.2 mmol/L combined with at least one of: oxygen saturation below 95% on room air, respiratory rate greater than 25 per minute, heart rate greater than 110 beats per minute, altered mental status, and temperature above 38.5ºC or below 35ºC.
Patients who trigger the sepsis alert are urgently (within 15 min) assessed bedside by a multidisciplinary team of physicians from the ED, the Department of Infectious Diseases, and the ICU, who establish preliminary diagnosis (assumed infection or not assumed infection), optimize clinical care, and decide level of care, i.e. ordinary ward, HDU, or ICU.
The present study was conducted between 27th of September 2017 and 31st of December 2018.
Consecutive patients who triggered the sepsis alert were subjected to structured sampling in the ED, including blood samples for CRP, PCT, neutrophils, lymphocytes, platelets, creatinine, and bilirubin. These samples were immediately analysed at the chemistry laboratory at the hospital, according to routine practice. In addition, blood was collected in a PPT ™ Plasma Preparation Tube (Becton Dickinson), which was immediately centrifuged for 10 minutes in order to separate plasma from blood cells, and then directly frozen at -18°C. After a maximum of 3 week, the samples were moved to -70°C. For all patients with written informed consent, the plasma samples were thawed and analysed for p-calprotectin, using a speci c particle enhanced turbidimetric assay by Gentian Diagnostics AS in Moss, Norway.

Data collection:
Data regarding demographic characteristics, comorbidity, physiological parameters, clinical, radiological, and microbiological data, and antimicrobial therapy were collected from the patient´s electronic records. Admission sequential organ failure assessment (SOFA) score was calculated from the rst physiological parameters registered (in the ambulance or ED) and the rst blood samples collected in the ED. Baseline SOFA score was determined by using the least pathological SOFA score parameters available within the time window, 7 days-90 days, prior to admission. If baseline parameters were missing, they were assumed to be normal, as in Seymour (24). Admission SOFA score minus baseline SOFA generated a delta-SOFA score.

De nitions of infection and sepsis
Sepsis alert patients were considered to have a bacterial infection if they received antibiotic therapy for 4 days or until death or until discharge, in line with de nitions suggested by the Centre for Disease Control (25). Patients were considered to have viral infection if a viral microorganism was veri ed by PCR or serology. Patients with bacterial or viral infection according to the de nitions above were considered to have sepsis if they had a delta-SOFA score of 2 or more on admission (26). Septic shock was de ned according to the Sepsis-3 criteria (26).

Primary outcome measures
The primary outcome was need of care in the ICU or HDU, which was decided by the multidisciplinary team.
Statistics SPSS statistical software (version 24) was used for comparison of proportions, using the chi-square test, and between-group comparisons, using the Mann-Whitney U test. STATA statistical software was used for receiver operating characteristic (ROC) curve analysis and comparison between areas under the ROC curves (AUC). A p-value of < 0.05 was considered signi cant.

Patient Characteristics
In total, 592 patients triggered the sepsis alert during the study period, of which 367 were included in the study. Three hundred and thirty-ve of these patients had an infection and 32 patients had no infection ( The patients with infection are described in Table 1, divided into those with or without direct transferral to ICU/ HDU. The groups had similar distributions of age, comorbidities, sources of infection, and 28-day survival rates. They had also similar rates of sepsis and delta-SOFA score, although septic shock was signi cantly more common in those with direct transferral to ICU/ HDU. Streptococcus pneumoniae 0 (0) 7 (8) Fungal 0 (0) 0 (0) *Data are presented in numbers (%), unless otherwise stated.

Performance of calprotectin and other biomarkers
The 367 sepsis alert patients in the study had the following median result: p-calprotectin 2.2 (IQR 1.1-3.8); CRP 67 (IQR 23-146); PTC 0.6 (IQR 0.2-6.0) NLR 11.4 (IQR 6.1-18.7). Figure 2 (A-D) shows the distribution of calprotectin, CRP, PTC and NLR in patients with infection with and without direct transferral to ICU/HDU, and patients without infection. Patients with infection and direct transferral to ICU/HDU had signi cantly higher p-calprotectin than patients with infection without direct transferral. No similar tendency was noted for PCT, CRP, or NLR. Figure 3 (A-D) shows the distribution of calprotectin, CRP, PTC and NLR among patients with bacterial infection, viral infection, and no infection. There was no signi cant difference for any of the biomarkers between the group with bacterial and viral infection. Only calprotectin could signi cantly distinguish between bacterial infection and no infection.   Figure 4 shows analysis of area under the curve (AUC) for need of ICU/ HDU care. When the analysis included all sepsis alert patients (Fig. 4A) and all patients with infections (Fig. 4B), p-calprotectin had signi cantly larger AUC than PTC and NLR and tended to have larger AUC than CRP. AUC-differences between calprotectin and the other biomarkers are shown in Table 3. As noted, AUC was signi cantly higher for calprotectin than the other biomarkers, both regarding all sepsis alert cases and regarding the patients with infection.

Discussion
In the present study of sepsis alert patients, we found that the novel biomarker p-calprotectin was elevated in patients with established infection and was superior to CRP, PCT, and NLR for detecting patients in need of ICU or HDU care.
Calprotectin has been shown to be a potential biomarker for discriminating sepsis patients from other patients with infection (16)(17)(18)(19). Due to the heterogeneity of sepsis patients, some researchers have focused on the role of calprotectin in multi-organ failure (27). We wanted to study calprotectin in a well characterized cohort of patients with clinical suspicion of sepsis already in the ED and evaluate whether calprotectin may be of value in the early assessment of sepsis. We found that patients with infection who the multidisciplinary team in the ED decided to transfer to the ICU or HDU had signi cantly higher calprotectin than other patients (Fig. 2). This was not explained by higher comorbidity (as measured by Charlson comorbidity score) or disease severity (as measured by delta-SOFA) on hospital arrival. There was no signi cant difference in delta-SOFA score between the group ending up direct in ICU or HDU and the group transferred to ward. This shows the di culties in discrimination patients with sepsis so severe that they need ICU care by only using delta-SOFA score. In this case the patients were also assessed by three different doctors. This also shows the need for better biomarkers, where calprotectin could have a place.
CRP, PTC, and NLR could not discriminate between those who were admitted to ICU/HDU and those admitted to wards, in line with ndings from similar studies (10,18,27,28). ROC curve analysis thus showed that calprotectin was superior to CRP, PCT, and NLR in assessing the need for ICU/ HDU care (Fig. 4, Table 4).
As shown in Fig. 4 (20). However, when looking at sensitivities, speci cities, and predictive values, we considered pcalprotectin 4.0 mg/L to be the best cut-off value for predicting the need for ICU/ HDU care, with a sensitivity of 42% and a speci city of 83%. Larsson et al. found the best p-calprotectin cut-off to be 1.3 mg/L for discrimination between sepsis and non-sepsis, with sensitivity 81% and speci city 56%, and 1.1 mg/L for prediction of ICU death, with sensitivity 80% and speci city 46% (20). Other studies have shown optimal calprotectin cut-offs of 3.4 mg/L for differentiating sepsis from post-operation in ammation (PPV47% and NPV 69%), e.g. in a study by Simm et al, and 2.2 (unknown unit) with sensitivity 62.5% and speci city 69.7% for blood culture positive sepsis in children in a study by Decambrinos et al. (16,19). This might be related to patient selection, analytic method for p-calprotectin or the nature of the study question.
P-calprotectin and the other biomarkers did not differ considerably in performance based on sex, age, comorbidity and source of infection and there was no clear difference in p-calprotectin levels between bacterial and viral infection. This was also the case for CRP, PCT, and NLR, and may be due to the small number of patients with viral infection in the present study. Also, to be noted, p-calprotectin was not higher among patients with veri ed bacteremia (positive blood culture), compared to those with negative blood cultures in our study (Table 2).
Faecal calprotectin has been shown to be affected by immune suppression (29), but we nd no difference in p-calprotectin levels in patients with or without immunosuppression.
On repeated samples, mostly NLR but also calprotectin decreased on day 2-3, whereas CRP and PTC increased, and had started to decrease on day 5-7. This may re ect different inherent kinetics of the biomarkers. Our ndings differ from those of Bartakova et al, and Simm et al (16,18). In Bartakova´s study all biomarkers hade the same dynamics as we found for calprotectin and NLR (18). However, in the study by Simm et al., only CRP had the same pattern as p-calprotectin (16). This may re ect different timing of sampling and a more heterogenous group of patients in our study compared with the others.
Since calprotectin increases early during infection, it could be potentially be useful in the pre-hospital care setting. Further studies will be needed to de ne its exact role.
The present study has several strengths. First, our study cohort was well characterized and already clinically strati ed for suspected sepsis, since it was part of a sepsis alert used in routine practice in the ED. All patients, even those who did not have any infection, could be assumed to be severely ill due to the inclusion criteria in the alert.
The study also has some limitations. For ethical reasons we did not try to obtain consent from relatives to patients who were already deceased. This was a concern for the 35 patients who died before consents were obtained, and we did not study how many of the deceased patients who were transferred to ICU or HDU before death. Thus, many severely ill patients were not included in the study and it is hard to assess how this might have affected the outcome of the study.
In conclusion, in sepsis alert patients, p-calprotectin was elevated in those who a multidisciplinary team decided to transfer immediately to the ICU/ HDU. P-calprotectin was superior to traditional biomarkers for predicting the need for ICU/ HDU care.
Conclusion P-Calprotectin was found to be superior to CRP, PCT, and NLR for identi cation of patients with suspected sepsis who were in need of immediate ICU or HDU care. Thus, P-Calprotectin appears to be a useful biomarker in the management of sepsis.
the capacity to do that. P-calprotectin could there for have a place in the emergency when working with sever sick patients.    Receiver operating characteristics analysis of calprotectin, CRP, PTC and NLR.