1 University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia 
2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 
Correspondence: 
Mitja Košnik, 
e: mitja.kosnik@klinika­golnik.si 

Key words: 
sepsis; cD 64 antigen; c-reactive protein; procalcitonin; neutrophil 

Cite as: 
Zdrav Vestn. 2017; 86:481–92. 
received: 19. 8. 2016 accepted: 19. 10. 2017 
DOi 10.6016/ ZdravVestn.2022 

Biomarkers to diagnose sepsis 
anja Žargaj,1,2 Peter Korošec,1franci Šifrer,1 Mitja Košnik1,2 
Abstract 

Background: Sepsis is a life-threatening organ dysfunction that arises when a host responds insuf­ficiently to an infection as bacteria enter the bloodstream. In recent years, SOFA scoring system has been used to identify poor organ functioning. Microbiological blood tests represent a gold standard in sepsis diagnostics. Reliable biomarkers for early detection of sepsis would greatly facilitate rapid and efficient treatment of sepsis. 
Methods: In a prospective non-interventional study we studied the diagnostic value of C-reactive protein (CRP), procalcitonin (PCT), neutrophil CD64 index, neutrophil granulocyte count and im­mature neutrophil count in patients with sepsis and patients with severe infection without sepsis. A total of 46 consecutive intensive-care-unit patients admitted for severe infection and 10 healthy controls were included. The patients were treated routinely according to the principles of good clini­cal practice. 
Results: Statistically significant differences between the two groups of patients have been established for the CD64 index, the PCT and the immature neutrophil count, whereas the differences in the CRP and the neutrophil granulocyte count are statistically non-significant. The highest diagnostic values were measured for the immature neutrophil count (AUC 0.91) and PCT (AUC 0.84). The combina­tion of biomarkers has been shown to have same predictive values as the immature neutrophil count and the PCT. 
Conclusions: The CD64 index was one of the less discriminating for drawing distinction between sepsis and severe infection without sepsis in intensive-care–unit patients. 
Cite as: Zdrav Vestn. 2017; 86:481–92. 


1. Background 
Sepsis is one of the commonest causes of death due to infection. In the USA hospitalizations for septicemia or sepsis increased from 621,000 in 2000 to 1,141,000 in 2008 (1). In Slovenia, the incidence of severe sepsis in adults is es­timated at 118 cases per 100,000 inhabit­ants (2). 
The sepsis mortality is between 17.4 % and 42.9 % (3). In developed countries mortality due to sepsis exceeds the mor­tality due to myocardial infarction (4). Sepsis is the most expensive condition treated in U.S. hospitals, costing more than $20 billion in 2011, and increasing on average by 11.9 % annually (5). 
A consensus conference in 1991 de­fined “sepsis” as the combination of an infection with two or more features of the so-called “systemic inflammatory response syndrome” (SIRS) (6): altered body temperature > 38 °C or < 36 °C, ele­vated pulse rate, elevated respiratory rate and abnormal white blood cell count 

12.0 × 109/L or < 4.0 × 109/L (7). However, this definition lacked specificity. There­fore, an updated definition defines sepsis as a systemic organ failure due to inap­propriate immune response to the incit­ing microorganism (8). 
For clinical operationalization, organ dysfunction can be represented by an in­crease in the Sequential [Sepsis-related] Organ Failure Assessment (SOFA) score of 2 or more points (Table 1) (9). 
Patients with suspected infection who are likely to have a prolonged ICU stay or to die in hospital can be promptly identified using qSOFA, i.e., al-
Table 1:sOfa score. (9). 

teration in mental status, systolic blood pressure . 100 mm Hg, or respiratory rate . 22/min (8,9). The mortality rate depends on the severity of illness. In sep­tic shock the hospital mortality exceeds 50 % (10,11). 
Septic shock is a subset of sepsis in which underlying circulatory and cel­lular/metabolic abnormalities are pro­found enough to substantially increase mortality (12). Patients with septic shock can be identified by persisting hypoten­sion requiring vasopressors to maintain MAP . 65 mm Hg and by the serum lac­tate level > 2 mmol/L (18 mg/dL) despite adequate volume resuscitation (8,13). 

Legend: PaO2 – Partial pressure of oxygen; FiO2 – Fraction of inspired oxygen; MAP – Mean arterial pressure; a – Catecholamine doses are given as µg/kg/min for at least 1 hour 
The gold standard for the diagnosis of sepsis is a microbiological examina­tion of blood. Whenever possible, two to four sets of blood specimens should be collected from different venipunc­ture sites. For most bacteria, the median time to positivity of hemoculture is 12 to 36 hours, whereas for certain bacte­ria and fungi this interval is even longer. When using traditional microbiologi­cal methods, further 48 to 72 hours are needed to identify the causative agent of sepsis and define its susceptibility to an­timicrobials (14-16). 
Treatment of sepsis has to be started early with broad-spectrum antibiotics, which is not only expensive, but also leads to multiple drug-resistant bacteria. Therefore, biomarkers with high pre­dictive value in the diagnosis of sepsis are needed (17). One of the earliest bio-markers used to diagnose infection is C-reactive protein (CRP), so named for its ability to precipitate from serum in the presence of pneumococcal cell wall C-polysaccharide. CRP is an acute-phase reactant found in the blood that is pro­duced by hepatocytes in the setting of infection or tissue injury. CRP produc­tion is triggered by cytokines (IL-1, IL-6 and TNF-.). Plasma CRP levels increase within 6–12 hours after an inflammatory stimulus and peak at around 48 h. It has a short half-life (16,18). 
Procalcitonin (PCT) is a precursor of the hormone calcitonin, which is pro­duced in the thyroid to regulate serum calcium concentrations. Under normal conditions, the thyroid gland is the only tissue that produces PCT and serum lev­els are very low (17). During infection, PCT is produced by other cells as well and its plasma levels increase. Although the exact stimuli that mediate PCT se­cretion are unknown, evidence suggests that early inflammatory signals such as TNF-., IL-1ß and IL-6 are involved (19). 
Elevations in PCT are generally observed before CRP rises and it peaks within a much shorter time frame. Additionally, when the patient responds to therapy, PCT levels return to normal much faster than CRP (20). 
CD64 is a neutrophil cell surface marker also known as Fc.R1 (21). It is the first of the three receptors on the neutrophil whose function is to bind the Fc portion of IgG (hence .) antibodies that facilitate bacterial opsonization and phagocytosis (22). CD64 is constitutively expressed on neutrophils, albeit at low levels, until the immune system encoun­ters an infectious agent whereupon the surface expression of CD64 is highly up-regulated (23). 
In the absence of stimulating factors, the CD64 expression decreases within 48 hours and normalizes within seven days (24). 
We aimed to compare the diagnos­tic accuracies of different biomarkers, namely C-reactive protein (CRP), pro-calcitonin (PCT), the neutrophil CD64 index, neutrophil granulocyte count and immature neutrophil count in the diag­nosis of sepsis. 


2. Methods 
We sought to enroll all admissions due to severe infection to the general intensive care unit (ICU) at the Uni­versity Clinic of Pulmonary and Aller­gic Diseases Golnik between January 2009 and February 2010. Ten healthy subjects served as controls. The study was approved by the Slovenian Medical Ethics Committee (reference number 63/05/04). All data were anonymized. Blood was collected from patients within six hours of their admission to the unit. 
Patients were treated according to good clinical practice. Most of the pa­tients received antibiotic therapy before admission to ICU. On admission, blood was drawn for routine blood tests (CRP, PCT, blood count) and additionally for the neutrophil CD64 index. In the analy­sis we included only those blood test results which were determined on the same day as the neutrophil CD64 index. Treating physicians were blind to the value of the neutrophil CD64 index. 

Patient records were examined to de­termine the clinical outcomes of hospi­talizations and assigned a clinical score denoting the likelihood of sepsis or bac­terial infection without sepsis. Thereaf­ter, the patients were assigned either to the sepsis group (positive blood cultures or the diagnosis of sepsis upon discharge from the ICU) or the infection without sepsis group. The CRP concentration was determined using the immunotur­bidimetric assay (Roche Diagnostics) on the Hitachi analyzer, and the PCT concentration by the electrochemilu­minescence method (Roche Diagnos­tics). CD64 expression on neutrophils was measured using the flow cytometer FACSCalibur (Becton Dickinson, NY, USA) and the Leuko64 kit (Trillium Di­agnostics, LLC, Maine, USA)., This test kit includes fluorescent beads and anti­bodies to CD64 and CD163. The patient’s sample provided both an internal nega-
Table 2:groups of patients. 
tive control (lymphocytes) and an inter­nal positive control (monocytes). The lymphocyte population was defined by forward and side scatter characteristics, and was distinct from granulocytes. Sur­face CD163 staining along with forward and side scatter characteristics were used to define the monocyte population. The neutrophil CD64 index was then calcu­lated using the ratio of the mean fluores­cent intensity of the cell populations to that of the beads. 
Continuous variables are presented as the mean ± SD for normally distributed data or the median (interquartile range (IQR)) for non-normally distributed data. Comparisons of group differences for continuous variables were performed by Mann-Whitney test. The p value of < 0.05 was considered to be statisti­cally significant. Categorical data were presented as the number of patients in each category with corresponding per­centages. The significance of differences in proportions was tested by Chi-square test. 
The performance of each biomarker for identifying sepsis was assessed as the area under a receiver operating charac­teristic (ROC) curve (AUC). For each biomarker ROC curves were used to de­rive cut-offs for sensitivity and specific-

Legend: *** – p<0.01; NS – statistically non-significant difference. 
ity to distinguish sepsis from infection without sepsis. 
The biomarker was highly discrimi­natory if the AUC was > 0.90 (25). 
The discriminatory value of a bio-marker was defined as the value higher than the highest value found in patients with infection without sepsis, and the minimal value as the highest biomarker value still excluding the diagnosis of sep­sis. 


3. Results 
We included 46 patients and 11 healthy controls (Table 2). 
In the group of sepsis there were 21 patients (47.6 % of men), the mean age was 70 (55, 76) years. Hemocultures were positive in 13 patients (62 %). The causes of infection were pneumonia in 14 patients ( in 11, in 1, in1, unknown in 1 patient), urinary infection in 4 patients ( in 3 patients, unknown in 1), endocar­ditis in 1 patient (, leg ulcer in 1 patient (, infection of the central venous canal in 1 patient (. All patients required ino-
Original scientific article 
tropic and vasopressor treatment. Seven patients had severe comorbidity (ad­vanced COPB in 2 and heart failure in 5 patients). Four (19 %) patients died. 
In the group of severe infection with­out sepsis there were 25 patients (52 % of men), the mean age was 68 (56, 77) years. The causes of infection were: pneumonia in 13 patients ( in 5, in 1, in 1, and unknown cause in 6 patients ), acute exacerbation of COPB due to different infections in 5 patients, empyema in 3 patients, tubercu­losis in 1, erysipelas in 1, acute bronchitis caused by in 1, and acute worsening of polymyositis due to respiratory infec­tion in 1 patient. In 17 patients severe co-morbidities were observed (COPB in 10, lung fibrosis in 2 and heart failure in 5 patients). Seven (28 %) patients died. 
There were no statistically significant differences by the gender ratio and age between the groups. In the group with infection without sepsis there was a sig­nificantly higher number (p < 0.05) of patients with severe comorbidities. Al­though more patients died in the group of severe infection without sepsis, the 

Table 3: Diagnostic accuracy of measured biomarkers in patients with sepsis compared to patients with infection without sepsis. 

Legend: Discriminatory value – value higher than the highest value found in patients with infection without sepsis; Minimal value 
– the highest biomarker value still excluding sepsis diagnosis in this study; Sensitivity – proportion of patients with sepsis with a biomarker above discriminatory value; ROC AUC – area under the ROC curve; 95 % CI – 95 % confidence interval. 
Biomarkers to diagnose sepsis on line edition 

Figure 1A–E: sepsis biomarkers in patients with sepsis (s) and in patients with infection without sepsis 
(O) (healthy controls (Z) used only for neutrophil cD64 index). 
Legend: Longer line shows the median, shorter line shows the interquartile range; ns – statistically non-
significant difference, **** – p<0.01; *** – p<0.05. 
difference was not statistically signifi­cant. 
The group of 11 healthy controls (55 % of men), was significantly younger com­pared to both groups of patients (mean age 32 (29, 35) years). 
The values of biomarkers in all three groups are shown in Figures 1A-E. 
Diagnostic accuracies of the mea­sured biomarkers are shown in Table 3. 
The biomarkers above the discrimi­natory value in the patients with sepsis are shown in Table 4. 
In 13 of the 21 patients, at least one biomarker was above the discrimina­tory value. In 10 patients more markers were above the discriminatory value at the same time, whereas in three patients only one marker was above the discrimi­natory value. Some patients did not have all markers determined on the same day as the CD64 index; those were excluded from the analysis. The combination of biomarkers was more likely to distin­guish sepsis from infection without sep­sis than a single marker. 
Table 4: Biomarkers above discriminatory value in patients with sepsis. 

4. Discussion 
In this study we compared the diag­nostic value of some biomarkers of in­flammation to establish the diagnosis of sepsis. We were particularly interested in the diagnostic accuracy of the neu­trophil CD64 index, which has been considerably studied in the diagnosis of sepsis in children (23,26) and in distin­guishing diseases that are clinically dif-


Legend: * – Biomarker above discriminatory value; nm – Not measured; Empty field – Biomarker below discriminatory value. 
ficult to distinguish between themselves, for example, sepsis from severe infection without sepsis. Therefore, we also di­vided the patients into two groups based on the clinical picture and the course of disease, the laboratory and the microbi­ological findings, into a group of sepsis and a group of infection without sepsis. 
In the sepsis group, similarly to other studies (22,27,28), we included patients with positive hemoculture and those with a proven infection, who were di­agnosed with sepsis by a physician, and patients who needed inotropic and va­sopressor support. Patients with severe infection without sepsis were predomi­nantly the patients with advanced lung and heart disease in whom the infec­tion further impaired the functioning of these organs. The incidence of signifi­cant comorbidities was statistically high­er in this group compared to the group of patients with sepsis. In the group of patients without sepsis, pneumonia was frequently caused by an influenza virus causing a severe course and high mor-
Table 5:summary of studies on diagnostic values of neutrophil cD64 index for sepsis amongst adults (21). 

tality. This can explain high mortality in patients with infection without sepsis. 
In our study, the serum CRP concen­tration in the patients with sepsis was not significantly higher than in the pa­tients with infection without sepsis. The AUC was 0.56 in the patients with sepsis compared to the patients with infection without sepsis, which means that the test had a little diagnostic value to discrimi­nate between both conditions. Most studies have proved that PCT is a better test for sepsis than CRP (29). Patil noted that serum procalcitonin concentrations in adult patients with positive hemo-culture were higher (8.66 ng/mL) than in patients with negative hemoculture 
(1.03 ng/mL) (p < 0.001) (30). Llewe­lyn summed up that the procalcitonin values were higher in severe infections, such as sepsis, than in patients with SIRS 
(3.1 ng/mL [0.8–3.9] versus 0.2 ng/mL [0.1–0.8] (p < 0.001) (18). In the analy­sis of patients in the intensive care unit, Gibot found that PCT distinguished pa­tients with sepsis from those admitted to the intensive care unit (p > 0.001) with a diagnostic AUC of 0.91 (31). 
On the other hand, PCT did not dis­tinguish sepsis from SIRS and other diseases in the study by Tanga and co­workers (32). Gibot also concluded that the concentration of PCT was higher in patients infected with Gram-negative 
30.09 ng/ml (4.55–76.01) than in those infected with Gram-positive organisms 1.33 ng/ml (1.04–4.98) (p < 0.0001) (31). 
In our study, the serum PCT con­centration was statistically significantly higher in the group of sepsis compared to the patients with infection without sep­sis. PCT (AUC 0.84) proved to be a bet­ter biomarker than CRP (AUC 0.56) in the diagnosis of sepsis, similarly to other studies (29-31). Also, PCT had a better diagnostic value for sepsis compared to the neutrophil CD64 index (AUC 0.78) 
and neutrophil count (AUC 0.69). The neutrophil CD64 index was expected to be significantly higher in both groups of patients compared to healthy controls, and was higher in the patients with sep­sis compared to the patients with infec­tion without sepsis, but in our patients its diagnostic accuracy was low (AUC 0.78). 
Although some publications have re­ported a high diagnostic value of this in­dex (33,34), many other studies, such as ours, have not confirmed it (35,36). One of the reasons for contradicting conclu­sions is the choice of patients in com­pared groups (Table 5). 
Our patients in the infection with­out sepsis group were internal medi­cine patients with severe comorbidities, clinically difficult to distinguish from sepsis. A similar result was obtained by Gamez-Diaz, who also included criti­cally ill patients requiring intensive care, and during hospitalization differenti­ated between critically ill patients with sepsis from those without sepsis (35). The studies that found a good diagnos­tic value of the neutrophil CD64 index included healthy subjects or patients af­ter surgery without infection in control groups (22,28,33). 
The CD64 test was expected to be the most discriminative (AUC was 1.0) when comparing the patients to healthy individuals. Certainly, to distinguish pa­tients with sepsis from healthy subjects we do not need laboratory tests. In clini­cal practice, however, it is necessary to distinguish among different diseases. 
The cut-off value where the test re­liably distinguished the patients with sepsis from the patients with infection without sepsis was 7.5, a high value, and many patients with sepsis had signifi­cantly lower values. However, the neu­trophil CD64 index had a good negative predictive value, since no patient with sepsis had this index below 1.2; the index of less than 1.2 certainly excluded sepsis in our study. 

In our study we did not confirm dif­ferences in neutrophil granulocyte count among the groups, whereas the concen­tration of immature neutrophil count was significantly higher in the patients with sepsis than in the patients with in­fection without sepsis. We found that the neutrophil CD64 index had a better diagnostic value than neutrophil granu­locyte count in the patients with sepsis compared to the patients with infection without sepsis. The highest diagnostic values were found for the immature neu­trophil count (AUC 0.91). 
So far, we have not found a biomark-er with sufficient (> 0.9) sensitivity and specificity to diagnose sepsis. However, an increasing number of studies (31,35) have indicated that combinations of var­ious biomarkers are a useful approach to improving the accuracy of diagnosing sepsis. In our study, at least one marker (CRP, PCT, neutrophil CD64 index, neu­trophil granulocyte count or neutrophil granulocyte count) was above the dis­criminatory value in 62 % of patients for distinguishing the patients with sepsis from those with infection without sepsis. A large majority of patients with sepsis were detected using only two markers, namely immature neutrophil count and PCT. 
Literatura 
The limitation of our study is het­erogeneity of the group of patients with infection without sepsis. Another limita­tion is that we assessed the performance of biomarkers in identifying sepsis and infection without sepsis only at the time of sampling. Thus, we cannot draw con­clusions about the predictive value of the investigated biomarkers for the potential later development of sepsis, or assess the impact of serial measurements. 

5. Conclusions 
The neutrophil CD64 index has proved to be one of the least discrimi­native biomarkers to distinguish sepsis from severe infection without sepsis in patients in the intensive care unit. The highest diagnostic values were provided by the immature neutrophil count (AUC 
0.91)
 and PCT (AUC 0.84). A combi­nation of biomarkers has been shown to have similar predictive values as the immature neutrophil count and PCT. However, the neutrophil CD64 index has proved to be useful regarding the nega­tive predictive value, since none of the patients with sepsis had the index below 

1.2.
The combination of biomarkers had better diagnostic accuracy compared to a single biomarker, whereas the most ac­curate biomarkers were the immature neutrophil count and PCT.


We would like to thank Julij Šelb, MD, PD, for helping with statistical analysis. 
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