a b s t r a c t

Objectives: To evaluate the value of presepsin in diagnosis and risk stratification of septic patients in emergency department, and investigate the utility in differentiation of gram-positive and Gram-negative bacterial infection. Methods: We enrolled 72 patients with sepsis and 23 nonbacterial patients with Systemic Inflammatory Response Syndrome who were admitted to the emergency department of Tianjin Medical University General Hos- pital. Meanwhile, 20 healthy volunteers were included. Plasma presepsin, serum PCT, C-reactive protein (CRP), lactate and white blood cells were measured, and APACHE II score were calculated upon admission. The receiver-operating-characteristic curve (ROC) was computed and the area under the ROC curve was for eval- uating the value to diagnose sepsis. Then the patients were grouped according to the result of culture and severity of sepsis.

Results: The levels of presepsin, PCT, CRP and WBC were apparently higher in sepsis patients than in nonbacterial SIRS group (P b 0.05). The levels of presepsin and the APACHEII score were demonstrated the significant differ- ence among sepsis, severe sepsis and septic shock patients (P b 0.05). The area under the ROC curve of presepsin, PCT, CRP and WBC were 0.954, 0.874, 0.859 and 0.723 respectively. The cutoff of presepsin for discrimination of sepsis and nonbacterial infectious SIRS was determined to be 407 pg/ml, of which the clinical sensitivity and specificity were 98.6% and 82.6%, respectively. Moreover, presepsin was significantly different between gram- positive and gram-negative bacterial infection (P b 0.05).

Conclusion: Presepsin was a promising biomarker for initially diagnosis and risk stratification of sepsis, and a potential marker to distinguish gram-positive and gram-negative bacterial infection.

(C) 2017

1. Introduction

Sepsis, defined as a systemic inflammatory response syndrome by infection, still represents a major cause of morbidity and mortality in critically ill patients, which can evolve to septic shock and multiple organ failure [1]. It was reported that sepsis mortality occurs in 65.5 per 100,000 persons in United States and the in-hospital mortality rate from sepsis is estimated to be 24.1% in Europe [2,3]. Alert and ear- liest timing of diagnosis and treatment are recommended as the best choice to prevent complex syndrome and high mortality. However, there is a great lack of evidence for biomarkers to reliably early diagnose and predict the future course of patients suffering from sepsis in the emergency department (ED) [4-6]. As such, interest has focused on bio- markers for early diagnosis, risk stratification, and evaluation of progno- sis of sepsis recently.

* Corresponding author.

E-mail address: [email protected] (S. Shou).

¹ These Authors equally contributed to the work.

Presepsin (Soluble cluster of differentiation 14 subtype, sCD14-ST) is a 13 kDa protein that is a truncated N-terminal fragment of CD14. It is released into circulation when monocyte is activated after binding with lipopolysaccharides (LPS) and LPS binding protein (LPB) in re- sponse to infection [7-10]. CD14 is a glycoprotein receptor expressed on the surface of various kinds of immune cells, such as monocytes, macrophages and neutrophils and releases soluble CD14 fragments (sCD14), which can also be secreted by hepatocytes. Besides, CD14 plays a crucial role in the activating the toll-like receptor 4 (TLR4), protecting against the invading microorganisms. Presepsin is the production of sCD14 cleavage with bacterial lysosomal enzymes and can be detected in the blood flow.

Although the blood culture is the gold standard of sepsis, it takes at

least 3 to 7 days with the low positive result. Moreover the positive rate of blood culture may further reduce after the antibiotic treatment. However, the early diagnosis and treatment of sepsis will decrease the mortality and morbidity. A potential biomarker to diagnose sepsis is es- sential. Pierrakos and Vincent has reported that there are N 178 septic biomarkers in 2010 [11], such as CRP, PCT, IL-27, suPAR etc. Maybe the number is increasing year by year. But no single biomarker can have

https://doi.org/10.1016/j.ajem.2017.12.038

0735-6757/(C) 2017

Table 1 Characteristics of subjects in this study
	Normal	SIRS	sepsis	Severe sepsis	Septic shock	Total
Case amount	20	23	33	24	15	115
Male	10(50%)	13(57%)	22(67%)	10(42%)	10(67%)	65(57%)
Female	10(50%)	10(43%)	11(33%)	14(58%)	5(33%)	50(43%)
Average age (range)	60.6	59.7	54.1	70.5	69.2	62.1
Origin	(18-85)	(18-92)	(18-100)	(18-92)	(24-95)	(18-100)
Pneumonia	-	-	15	16	11	42
Hepatobiliary	-	-	3	5	-	8
Gastrointestinal	-	-	1	1	2	4
Urinary	-	-	6	1	1	8
CNS	-	-	1	-	-	1
Others	-	-	7	1	1	9
Category Gram positive	-	-	4(12.1%)	2(8.3%)	2(13.3%)	8
Gram negetive	-	-	5(15.2%)	6(25.0%)	7(46.7%)	18
Fungus	-	-	3(9.1%)	3(12.5%)	1(6.7%)	7
Mixed (bacterial and fungus)	-	-	2(6.0%)	6(25.0%)	0(0.0%)	8
Unknown	-	-	19(57.6%)	7(29.2%)	5(33.3%)	31

the sufficient sensitivity and specificity. Presepsin is a specific and promising biomarker of sepsis, which has been used for the early diagnosis and prognosis of sepsis. Nevertheless, whether presepsin can risk stratification of sepsis and play a vital role in distinguishing the gram-positive and gram-negative infection are still unclear. There- fore we decide to perform a study to the focus in detail.

1. Material and methods
   1. Samples collection and measurement

The study was permitted by Tianjin Medical University General Hos- pital ethics committee. We collected the blood samples of the patients admitted to the emergency department at Tianjin Medical University General Hospital after patient’s agreement. The patients were classified into two groups: sepsis and non-infectious SIRS. Sepsis patients were diagnosed according to the guideline of American College of Chest Phy- sicians/Society of Critical Care Medicine (ACCP/SCCM), and then divided into sepsis, severe sepsis and septic shock groups by the severity. Based on the infectious origin, the septic patients were divided into pneumo- nia, hepatobiliary, gastrointestinal, urinary, CNS and others. Based on the category of infectious microbe, the septic patients were further classified into Gram-positive bacteria, gram-negative bacteria, mixed (bacterial and fungal), fungus and unknown. Meanwhile, 23 non- infectious SIRS patients and 20 healthy volunteers were included. Patients who had autoimmune disease, immunodeficiency disease, tumor and immunosuppressive therapy recently were excluded.

Measurement and methods

We measured the presepsin, PCT, CRP and WBC in blood specimens collected upon admission. The peripheral venous blood samples were collected before treatment, especially the antibiotic treatment.

(1) Presepsin assay: presepsin concentrations were measured by the

PATHFAST analyzer, based on the chemiluminescent enzyme immuno- assay (Mitsubishi Corp, Japan) with the peripheral venous blood in the EDTA tube. (2) Lactate assay: the arterial blood was collected and mea- sured by GEM premier 3000 Blood gas analyzer (IL Corp, USA). (3) PCT, CRP and WBC were measured by the clinical laboratory of Tianjin med- ical university general hospital.

Statistical analysis

Statistical analysis was carried out using the SPSS version19.0. The data was all tested by normal distribution test. Mean +- SD value was used to analyze data with normal distribution. Comparison between two groups was performed using least significant different (LSD-t) test and multiple comparisons were performed using the ANOVA analysis. The median (with inter-quartile rang) was used to analyze data with abnormal distribution. Two-group comparison was performed using Mann-Whitney U test and multiple comparison was performed using Kruskal-Wallis H test. The receiver-operating characteristic curve was analyzed to evaluate the diagnostic properties of septic biomarkers, in- cluding presepsin, PCT and others.

1. Results
   1. Subjects characteristics

Characteristics of the 115 subjects were shown in Table 1, which contains 65 males and 50 females. The average age of 115 patients was 62.1 (18-100) years old in the study. The average ages of normal, SIRS, sepsis, severe sepsis and septic shock group were also shown in Table 1. Two variables (gender and average age) were found no statisti- cally significant difference among each group. The infection origins were various, including pneumonia, hepatobiliary, gastrointestinal, uri- nary, CNS and others.

Table 2

Comparison of clinical data in different groups with the severity of sepsis

Group	N	APACHEII score		Presepsin(pg/ml)		PCT (ug/L)		Lactate (mmol/L)
		Mean +- SD		Mean +- SD		Median(min,max)		Median(min,max)
Sepsis	33	13.73 +- 4.118		719.97 +- 215.890		0.80(0.05,13.67)		1.00(0.2,5.1)
Severe sepsis	24	22.17 +- 6.329?		1421.21 +- 643.182?		2.94?(0.19,76.57)		2.0?(0.5,6.9)
Septic shock	15	26.47 +- 8.026?		2564.13 +- 1557.556?		3.46(0.33,150.41)		2.6(0.8,10.5)
Statistics		29.046?		27.098?		12.685		18.891
P value		0.000		0.000		0.002		0.000

Statistics ^? mean F value, other statistics mean ?² value; compared with sepsis group, ^?P b 0.05;compared with severe sepsis, ^?P b 0.05.

Fig. 1. The area under the ROC curve with different parameters in septic patients.

The characteristics of the infectious origin and category of infectious microbe in different groups were all shown in Table 1.

Comparison of sepsis and non-sepsis (including normal and SIRS group) patients

The median level of presepsin was 965 pg/ml (range: 656.3-1665.5) in sepsis group (n = 72), 231 pg/ml (range: 234-393) in Normal group (n = 20), 298 pg/ml (range: 234-393) in SIRS group (n = 23). The presepsin level of sepsis group was higher than the normal group and SIRS group (U = 105, P b 0.01 and U = 76, P b 0.01, respectively). There was a significant difference between healthy control and non- infectious SIRS group (U = 97, P b 0.05).

Comparison of clinical data in septic patients according to the severity of sepsis

There was significant difference in presepsin, PCT, lactate and APACHEII score among sepsis, severe sepsis and septic shock group (P b 0.05). The levels of presepsin, PCT, lactate and APACHEII score in se- vere sepsis group were clearly higher than sepsis group (U = 94, 220, 107, 178, respectively, P b 0.05). Compared with severe sepsis group, the levels of presepsin, APACHEII score and lactate were significantly higher in septic shock group (U = 154, 125, 143, respectively, P b 0.05)(Table 2).

ROC analysis of the inflammatory markers in sepsis

ROC analysis revealed an optimized cutoff value of 407 pg/ml for presepsin and demonstrated a high Discriminatory power (AUC = 0.975) (Fig. 1).The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 98.6%, 90.7%, 94.67% and 97.48% respectively. Presepsin was a better biomarker in diagnosing sepsis compared with the other markers (Table 3).

Comparison of inflammatory markers in septic patients infected various kinds of pathogens

No statistically significant difference was found for presepsin, CRP, PCT and WBC in patients with gram-positive, gram-negative, mixed (bacterial and fungal), fungal infection and unknown infection (P = 0.133, 0.452, 0.499 and 0.984, respectively) (Table 4). However, the level of presepsin between gram-positive and gram-negative infection showed significantly different (P b 0.05), while PCT, CRP and WBC did not (Table 5).

1. Discussion

Nowadays lots of single or combined biomarkers were used to diag- nose and predict mortality in sepsis [12,13], including PCT, CRP, inter- leukin, soluble urokinase plasminogen activator receptor (suPAR) etc. But most of them can not play a vital role in sepsis. PCT was widely stud- ied, and its level also elevates in non-infectious SIRS patients, suffered from severe trauma, extensive burns, pancreatitis, organ transplanta- tion and invasive surgery [5,14]. Mata-analysis had demonstrated that the discriminatory power of PCT was low, both of the sensitivity and the specificity only 71% (95%CI:67%-76%) [15]. Single PCT is unable to discriminate sepsis from critical disease, which might need to accompa- ny with other Diagnostic biomarkers. Although the blood culture is the best reference in diagnosis and treatment, the low positive rate, lower

Table 3

The AUC of various indicators in patients with sepsis and nonbacterial SIRS Inflammatory parameter AUC SE P value 95%CI

Presepsin	0.954	0.023	0.000	(0.910,0.998)
PCT	0.874	0.041	0.000	(0.793,0.955)
CRP	0.859	0.039	0.000	(0.782,0.936)
WBC	0.723	0.060	0.001	(0.606,0.840)

Table 4

Comparison of clinical data in groups with various infected pathogens

Group	N	WBC(x109/L) Mean +- SD	Presepsin(pg/ml) median(min,max)	PCT(ug/L) median(min,max)	CRP(mg/dL) median(min,max)
Gram-positive bacteria	8	15.15 +- 5.69	1070(356,5844)	1.13(0.1150.4)	7.55(2.33,15.0)
Gram-negative bacteria	18	14.47 +- 7.73	1128(545,3890)	2.01(0.2,71.2)	11.60(1.25,29.7)
Mixed bacteria and fungus	8	15.74 +- 3.23	1757(420,2373)	3.41(0.1,47.1)	8.85(0.64,30.5)
Fungus	7	13.66 +- 9.75	922(443,3074)	2.35(0.1,76.6)	11.3(3.00,18.2)
Unknown	31	114.74 +- 7.01	743(470,4828)	1.20(0.1,17.7)	8.2(0.33,31.50)
Statistics		0.094?	7.053	3.366	3.672
P value		0.984	0.133	0.499	0.452

Statistics ^? mean F value, other statistics mean ?² value.

than 10% [16], and the long measurement period dramatically effected its efficiency and application in clinic. In reality, the physicians have to diagnose and treat sepsis patient relying on their own experience. Therefore, a high sensitivity and specificity with high effectiveness and efficiency index is imperative.

Presepsin, a new biomarker, was discovered to diagnose sepsis in 2004 [17]and was closely related with monocyte-macrophage activation in response to infection [18]. The level of presepsin was low in the serum of healthy individuals and began to increase within 6 h in peripheral venous blood after the onset of infection, which was earlier and faster than PCT and CRP. In our study, the levels of presepsin in septic patients were significantly higher than in non-infectious SIRS patients and healthy volunteers (P b 0.05), and were also significantly different between gram-positive and gram-negative infection (P b 0.05), which was in concordant with the studies [19]. The ROC curve analysis revealed the discriminatory power of sepsis, including presepsin, CRP, PCT and WBC. Our findings indicated that AUC for presepsin was better with higher sensitivity and specificity (98.6% and 90.7%, respectively), similar with Shozushima’s and Liu’s report [8,20]. All results demonstrated presepsin was a better biomarker for the diagnosis of sepsis and severe sepsis compared to PCT. On the contrary, Ulla et al. had reported the AUC of presepsin was lower than PCT in septic patients [21].

The cut-off value of presepsin in our study was 407 pg/ml. Similar was proven by Matej Godnic et al. with the cut-off of 413 pg/ml for di- agnosing sepsis [22]. However, a cut-off of 600 pg/ml was demonstrated by Endo et al. [19]. With the optimal sensitivity and specificity for diag- nosing the bacterial infection, we evidenced that presepsin was induced at the early stage of sepsis and played a more sensitive and specific role in diagnosing sepsis, compared with PCT, CRP and WBC.

The levels of presepsin increased significantly with the severity of sepsis and were found significant difference among sepsis, severe sepsis and septic shock groups. Carpio R et al. obtained similar results in the research [23]. Previous studies revealed that presepsin was elim- inated from the blood by renal catabolism [24]. Meanwhile, acute kid- ney injury occurred commonly in the progress of sepsis, which might be a underlying factor for the increasing presepsin, together with the se- verity of sepsis [25]. However, the levels of presepsin between sepsis

and septic shock group were no difference in Matej Godnic’s report [22]. Ulla et al. had also suggested that the concentration of the presepsin was not related to the severity of sepsis in the very first hours [21].

The level of presepsin will rise significantly in the early-onset sepsis. Whether presepsin can indicate the Gram-positive and Gram-negative bacterial infection is still controversial. We have demonstrated that presepsin can distinguish Gram-negative infection from Gram-positive infection. Presepsin is a receptor of LPS, which is one of the components for the Gram-negative bacterial cell wall. Presepsin is needed to activate endothelial and epithelial cells by LPS. Besides, Serge Masson et al. reported that patients infected with Gram-negative had higher levels of presepsin than those infected with Gram-positive [26]. However, some have reported that there was no significant difference between Gram-positive and Gram-negative bacterial infections [9,19, 27].

The rapid diagnosis and treatment play a vital role in improving the prognosis in patients with sepsis. It takes only 17 min to measure presepsin and can be tested at bedside, which is fast, convenient and financial accessibility, complying with the recommendation of the international guidelines for management of severe sepsis and septic shock:2012 [28].

Conflict of interest

None.

Acknowledgement

We thank professor Shu-Zhang Cui of Emergency Department of Tianjin Medical University General Hospital for the guidance of experi- mental design.

Funding

This work was supported by grants from the Tianjin Health Bureau Science and Technology funding (Nos. 2015KZ114) and the TMUGH funding (Nos. ZYYFY2016026).

Table 5

Comparison of clinical data in groups between Gram-positive and Gram-negative bacterial infection

Group	N	WBC(x109/L) Mean +- SD	Presepsin(pg/ml) median(min,max)	PCT(ug/L) median(min,max)	CRP(mg/dL) Mean +- SD
Gram-positive bacteria	8	14.8 +- 2.7	871(556,1077)	0.5(0.06,1.05)	8.3 +- 2.8
Gram-negative bacteria	18	14.1 +- 7.6	1448(554,3890)	0.53(0.1,45.52)	9.0 +- 7.1
Statistics		3.684?	7.053	3.366	3.498?
P value		0.067	0.013	0.177	0.074

Statistics ^? mean F value, other statistics mean U value.

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