Usefulness of neutrophil-to-lymphocyte count ratio, procalcitonin, and interleukin-6 for severity assessment of bacterial sepsis

Objectives: To explore the usefulness of neutrophil-to-lymphocyte count ratio (NLR), procalcitonin (PCT), and interleukin-6 (IL-6) for the severity assessment of bacterial sepsis. Methods: This study enrolled 100 patients with bacterial sepsis (disease group) who presented to Jinhua Central Hospital between March 2022 and March 2023 and 90 healthy individuals (control group). The patients were categorized into sepsis (64 cases), severe sepsis (18 cases), and septic shock (18 cases) groups according to the disease severity. The groups were compared in terms of the NLR, PCT, and IL-6, as well as the usefulness of these parameters, both alone and in combination, for the severity assessment of bacterial sepsis. Results: The NLR, PCT, and IL-6 levels were signi ﬁ cantly di ﬀ erent among the three groups, with increasing values corresponding with disease aggravation. The area under the curve (AUC) values of the combinations of NLR, PCT, and IL-6 levels were higher than those of single markers. The sensitivity and AUC value of the combination of PCT and IL-6 levels were the highest (0.87), with a similar AUC value of the combination of NLR, PCT, and IL-6 (0.865); however, the speci ﬁ city was signi ﬁ cantly improved with the latter (0.938 vs. 0.859). Conclusions: NLR, PCT, and IL-6 levels are signi ﬁ cantly increased in bacterial sepsis, and the combination of PCT, and IL-6 levels can improve the sensitivity of the evaluation ability for severe sepsis, and is more economical.


Introduction
Sepsis is a systemic inflammatory response syndrome mainly caused by trauma and infections.The disease severity is categorized as sepsis, severe sepsis, and septic shock.Sepsis has high morbidity and mortality rates.Recent epidemiological data indicate that approximately 30 million patients develop sepsis annually worldwide, with a mortality rate as high as 25 % [1,2].As patients with severe sepsis and septic shock are often accompanied by organ failure or irreversible hypotension, their mortality is much higher than that of patients with common sepsis.Therefore, early identification of the severity of infections and timely administration of standardized treatment can significantly improve the prognosis of patients with sepsis [3].
Routine blood tests are often used as indicators of the diagnosis and prognosis of sepsis in clinical practice, but white blood cell count (WBC>12 × 10 9 /L or <4 × 10 9 /L indicates inflammation) alone does not accurately reflect the clinical condition.Neutrophil to lymphocyte count ratio (NLR) is a new inflammatory marker calculated by using blood routine results.It is used as a sensitive indicator to evaluate inflammatory response, and is currently widely used in the prognosis judgment of infectious diseases [4], acute coronary syndrome [5], malignant tumors [6] and other diseases.A retrospective cohort study showed that NLR could be used as a predictor of in-hospital mortality in sepsis patients [7].In addition, studies have shown that NLR can accurately reflect the severity of sepsis [8].Procalcitonin (PCT) is a widely used inflammatory marker in clinical practice.It has good sensitivity to identify bacterial infection.When the body is attacked by bacteria, its concentration can start to rise sharply in 2∼3 h and reach a peak in 12∼24 h.However, the specificity of this marker is not high, and non-infectious states such as tumors and autoimmune diseases can also lead to its increase [9].As a recent study showed, there was no statistically significant difference in procalcitonin (PCT) levels in the sepsis group compared to the group with other non-inflammatory conditions, including trauma, ketoacidosis, hyponatremia, stroke, gastrointestinal perforation bleeding, and cardiac arrest [10].Interleukin-6 (IL-6) is the "vanguard" in the inflammatory response.Its concentration level rises rapidly after infection, can reach a peak in about 2 h, and the half-life is only 1 h, can quickly reflect the therapeutic effect, mainly used as an early diagnostic and prognostic marker of acute infection [11].In this study, we investigated the usefulness of the three most commonly used clinical indicators for evaluating the severity of bacterial sepsis, which can be used for the early identification and treatment of critically ill patients with sepsis to improve the prognosis.

Participants
This study included 100 patients (58 males and 42 females; mean age: 66.38±14.37years) with bacterial sepsis who fulfilled the diagnostic criteria for sepsis (sepsis 3.0) and were admitted at Jinhua Municipal Central Hospital between March 2022 and March 2023.The inclusion criteria were as follows: bacterial growth in blood culture; Sequential Organ Failure Assessment (SOFA) score ≥2; and informed consent provided by patients and/or their families.The exclusion criteria were as follows: children, pregnant women, and lactating women; SOFA score <2; major trauma, major surgery, myocardial infarction, cardiogenic shock, and pancreatitis; and no antibiotics received before admission.In addition, 90 healthy individuals (mean age: 66.11±12.61years) were selected as the control group, including 50 males and 40 females.The 100 patients with sepsis were categorized into the sepsis group (n=64; 38 males and 26 females; mean age: 66.59±14.55years), severe sepsis group (n=18; 10 males and eight females; mean age: 67.39±12.25 years), Severe sepsis group (n=18; 10 males and eight females; mean age: 66.59±14.55years), and septic shock group (n=18; 10 males and eight females; mean age: 67.39±12.25 years).The mean age of the participants was 64.61±15.11years.There were no significant differences in age or gender between the groups (p>0.05).

Methods
In total, 6 mL of venous blood was collected from patients with bacterial sepsis before the initiation of antibiotic treatment.Then, 2 mL of blood (EDTA-K2 anticoagulant) was subjected to routine blood tests on an XS-800i hematology analyzer (Sysmex Company, Japan), whereas 4 mL of blood (heparin anticoagulant) was used to detect PCT and IL-6 on a Pylon immunoassay instrument (Suzhou Star Child Medical Technology Co., Ltd.).

Statistical analysis
SPSS 22.0 software was used for statistical analysis.Data with normal distribution are presented as mean±standard deviation.Student's t-test was used for comparisons between two groups, and analysis of variance was used for comparisons between multiple groups.Data with non-normal distribution are presented as median ([P25, P75] quartile).Groups were compared using the non-parametric rank sum test.Count data are expressed as rates, and groups were compared using χ 2 tests.Receiver operating characteristic (ROC) curve was used to evaluate the efficacy of NLR, PCT, and IL-6 alone and in combination for the detection of critical sepsis (severe sepsis + septic shock).p-Value <0.05 was considered statistically significant.

Basic information of patients
Among the 100 bacterial sepsis patients included in this study, 38 strains (38 %) of Gram-positive bacteria were detected, of which the top three were Staphylococcus epidermidis (nine strains), Staphylococcus hominis (seven strains), and Staphylococcus aureus (six strains).A total of 62 strains of Gram-negative bacteria were detected, among which the top three were Escherichia coli (28 strains), Klebsiella pneumoniae (21 strains), and Acinetobacter baumannii (three strains).Among 100 patients with sepsis, eight died, corresponding to a mortality rate of 8 %, including two deaths in the sepsis group (3.1 %, 2/64), two in the severe sepsis group (11 %, 2/18), and four in the septic shock group (22 %, 4/18).

Comparison of blood routine parameters between the sepsis and healthy control groups
Compared with the healthy control group, the levels of WBC, N%, and NLR in the bacterial sepsis group were significantly increased, while L% was significantly decreased (Table 1).

Comparison of Gram-positive and Gramnegative bacteria among sepsis, severe sepsis, and septic shock groups
Twenty-six strains of Gram-positive bacteria and 38 strains of Gram-negative bacteria were detected in the sepsis group (n=64).Seven strains of Gram-positive bacteria and 11 strains of Gram-negative bacteria were detected in the severe sepsis group (n=18).Five strains of Gram-positive bacteria and 13 strains of Gram-negative bacteria were detected in the septic shock group (n=18).There were no statistically significant differences in the proportion of Gram-positive and Gramnegative bacteria among the three groups (p>0.05)(Table 2).
Comparison of hospital stay, routine blood parameters, and PCT and IL-6 levels among sepsis, severe sepsis, and septic shock groups Compared with the sepsis group, the PCT and IL-6 levels in the severe sepsis group were significantly increased; the levels of N%, NLR, PCT, and IL-6 in the septic shock group were significantly increased; and the hospital stay duration and L% were significantly decreased.Compared with the severe sepsis group, the NLR, PCT, and IL-6 levels in the septic shock group were significantly increased, and the hospital stay duration was significantly decreased (Table 3).

Logistic regression analysis for sepsis severity
Patients with severe sepsis and septic shock were included in the critical sepsis group.Logistic regression analysis was performed for the hospital stay duration, N%, L%, and NLR, PCT, and IL-6 levels.The results showed that the odds ratios for NLR, PCT, and IL-6 levels were 1.119, 1.042, and 1.002, respectively.There were no significant differences among the groups in terms of the other indicators (p>0.05)(Table 4).

Discussion
Sepsis is an organ dysfunction syndrome mainly caused by trauma and infections, which is mainly characterized by chills, high fever, and mental state changes.In severe cases, multiple organ dysfunction syndrome, diffuse intravascular coagulation, and even shock can occur.Bacterial sepsis is one of the most common types of sepsis.The incidence of bacterial sepsis is increasing due to worsening bacterial resistance, more frequent application of traumatic diagnostic and treatment technologies, and host immune deficiency.Although numerous recent studies have shown that the detection rate of Gram-positive bacteria is increasing, Gram-negative bacteria are still the main type of infectious bacteria [12].Consistent with these findings, in our study, the prevalence of Gram-negative bacteria (62 %) was higher than that of Gram-positive bacteria (38 %).There were no significant differences in the distribution of Gram-positive and Gram-negative bacteria among the sepsis, severe sepsis, and septic shock groups.Gram-negative bacteria were the main cause of infection in all three groups.Further observation showed that Gram-negative bacteria accounted for a higher proportion (5/8, 62.5 %) of the eight patients who died than Gram-positive bacteria (3/8, 37.5 %), which is consistent with a meta-analysis showing that sepsis caused by Gramnegative bacteria has a higher mortality rate [13].
Sepsis is associated with high morbidity and mortality rates worldwide [1,2].A national cross-sectional study conducted in 2020 revealed that there were 20.6 sepsis among 100 patients, with a 90-day mortality rate of 35.5 % [14].Similar epidemiological data show that patients with critical sepsis account for about 6-54 % of ICU patients, with a mortality rate of 20-60 %.Even if patients survive, their quality of life is significantly reduced, which shows the huge negative impact of critical sepsis on society [1].In this study, severe sepsis accounted for 36 % of sepsis, and although the overall mortality rate with bacterial sepsis admitted to the ICU was 8 %, and the mortality rate was as high as 22 % in the septic shock group.This high mortality rate may be due in part to delayed recognition and management of critical sepsis, with one study showing that septic patients who received anti-infective treatment within 1 h had a 33 % reduction in mortality [15].However, misuse of antibiotics imposes an additional financial burden on patients as well as increases the risk of drug resistance.Therefore, early and accurate assessment of sepsis severity and the development of reasonable and effective treatment plans are essential.
Sepsis is characterized by imbalance in the body's inflammatory response.When bacteria invade the blood, a large number of neutrophils are released from the bone marrow into the blood, participating in the processes of phagocytosis and sterilization.In addition, due to immune response overstimulation, lymphocytes secrete antiinflammatory factors, such as IL-10, to inhibit the inflammatory response, which leads to the marginalization and redistribution of lymphocytes, thereby reducing the number of lymphocytes.In this study, WBC, N%, and NLR were significantly higher in the bacterial sepsis group than  in the healthy control group, whereas L% was significantly lower in the bacterial sepsis group than that in the healthy control group.NLR is the ratio of neutrophil count to lymphocyte count.Because this parameter is calculated, economical, simple, and accurately reflects the activation and regulation of inflammation in the body, it has recently become a research hotspot.A prospective study showed that NLR was increased in the sepsis group, particularly in the septic shock group, and it was significantly correlated with the sepsis severity assessed by the SOFA score [16].Zahorec et al. found that the NLR was increased in deteriorating patients with sepsis who were admitted to the ICU, and the mortality rate was increased to 64 % with NLR>11.9 [4].In this study, NLR was significantly different among the sepsis, severe sepsis, and septic shock groups, and NLR was increased with disease severity.According to the ROC curve analysis, the optimal cut-off value of NLR was 10.29, i.e., NLR>10.29 accurately indicated the presence of critical bacterial sepsis.PCT is a precursor of calcitonin, a glycoprotein containing 116 amino acids that helps identify bacterial and viral infections [17].When bacteria invade the blood, large quantities of PCT are released through the direct pathway associated with lipopolysaccharides or the indirect pathway related to inflammatory factors [18].Clinicians often decide whether to stop or replace antibiotics based on changes in PCT level [19].A multi-center study categorized 266 patients with sepsis into the experimental and control groups.In the experimental group, antibiotic treatment was guided by the PCT level.The results showed that the sequelae and mortality were significantly reduced in the experimental group [20].Chen et al. found that PCT levels at 24 h after admission were predictive of 28-day survival in patients with sepsis, with a 28-day survival rate of 93.4 % in those with a low PCT level (<2.85 ng/mL) and 51.7 % in those with a high PCT level (>2.85 ng/mL).However, this marker has not been used to assess the sepsis severity [21].The AUC of PCT was higher than that of NLR (0.74 vs. 0.677).Compared with NLR, PCT is a superior biomarker for predicting the severity of bacterial sepsis.However, the use of PCT also has certain limitations because several conditions, such as tumors, autoimmune diseases, and other non-infectious diseases, can lead to an increase in the PCT level, resulting in a decrease in its specificity [9].IL-6 is a newly developed inflammatory marker for use in clinical laboratories.It is recognized as the "vanguard" of the inflammatory storm.When the body is attacked by bacteria, T cells and macrophages release large quantities of IL-6, which is mediated by glycoprotein 130.Then, IL-6 binds to pattern recognition receptors (such as Toll-like receptors), triggering an inflammatory cascade, which results in the production of a large number of inflammatory factors [22].The IL-6 level often increases earlier than PCT and can reach the peak level within 2 h after infection.A higher IL-6 level is associated with higher severity and mortality of sepsis [23].At present, it is mainly used for the early diagnosis of acute infections, evaluation of disease severity, and prognostication [11].In this study, the IL-6 level was significantly different among the three groups and was increased with disease aggravation.IL-6 level >581.61pg/mL indicates the presence of critical bacterial sepsis.
Because the occurrence and development of sepsis are complex processes, the use of a single indicator to evaluate the severity of bacterial sepsis does not achieve the ideal sensitivity and specificity levels.Zeng et al. evaluated multiple indicators related to sepsis and found that the combination of any two indicators can improve the sensitivity and specificity for the diagnosis of sepsis [24].Zeng et al. selected the most common clinical indicators (NLR, PCR, and IL-6) for the ROC curve analysis.The results showed that, compared with NLR, PCT, and IL-6 levels alone, the AUC value of pairwise combination for evaluating the severity of critical bacterial sepsis was increased, and the AUC value (0.87) and sensitivity (0.8) of PCT combined with IL-6 was the highest.The AUC value of the combination of the three indicators for evaluating critical sepsis was 0.865, although it was slightly lower than that of PCT combined with IL-6.Furthermore, the specificity was significantly improved after the addition of NLR, which can reduce the fluctuations in levels due to non-infectious factors, such as trauma, tumor, and autoimmune diseases.However, from the perspective of economics, the combined PCT and IL-6 model is more economical and simple, can reduce the medical expenses of patients, and is more sensitive, which is conducive to clinicians to make treatment plans as soon as possible.Therefore, this study recommends the use of the combined PCT and IL-6 model to assess the severity of sepsis.However, this study has certain limitations.First, this was a retrospective study, making it prone to selection bias.Second, only the initial values of inflammatory markers of hospitalized patients were collected, which revealed certain differences in the infection process of different patients.Third, only a small number of clinical cases were included, particularly in the severe sepsis and septic shock groups.A larger number of clinical cases need to be included to improve the reliability of results.

Conclusions
Bacterial sepsis is an important public health problem, especially critical sepsis, which has a huge negative impact on society, and it is urgent that we can quickly and accurately identify high-risk groups in clinical.As common markers for clinical development, NLR, PCT and IL-6 can be used for the early diagnosis of bacterial sepsis and as effective markers for assessing the severity of sepsis.The combination of PCT (>13.68) and IL-6 (>581.61)can improve the sensitivity of diagnosing critical sepsis.Clinicians can use the levels of each index to formulate timely treatment plans, thereby improving the prognosis of patients and reducing the mortality rates.In addition, due to the higher mortality rate of sepsis patients infected with Gram-negative bacteria, we should pay attention to sepsis caused by these bacteria and treat it as early as possible.

Table  :
Comparison of routine blood tests between the bacterial sepsis and healthy control groups.

Table  :
Distribution of Gram-positive and Gram-negative bacteria in the sepsis, severe sepsis, and septic shock groups.

Table  :
Logistic regression analysis for sepsis severity.

Table  :
Comparison of NLR, PCT, and IL- levels for the diagnosis of critical sepsis.

Table  :
Comparison of hospital stay, routine blood parameters, and PCT and IL- levels among sepsis, severe sepsis, and septic shock groups.Compared with sepsis group, a p<.;Compared with severe sepsis group, b p<..