Impact of Neutrophil Gelatinase-associated Lipocalin A Novel Sepsis Biomarker on PRISM III Score in Predicting Sepsis Severity And Mortality in Pediatric Intensive Care Units (Single Center Study)

doi:10.21203/rs.3.rs-3569764/v1

Background: Physiological, pathological, and metabolic problems brought on by infection are what define the syndrome of sepsis. Neutrophil granules generated at human sites of infection and inflammation were the source of the first discovery of neutrophil associated gelatinase associated lipocalin (NGAL).

Objectives: to evaluate the predictive usefulness of serum NGAL in children with sepsis in the pediatric intensive care unit and to compare it with the clinical score (PRISM III score) that is most frequently used to predict mortality in critically unwell children. ).

Patients and methods: In this study 75 patients were included according to the inclusion criteria and divided into three groups 25 each (sepsis, septic shock, and multi-organ failure), and serum NGAL level in those group was measured and compared to 25 healthy controls.

Results: Serum NGAL was significantly higher in both septic shock and multi-organ failure groups than in the control group. Positive bacterial cultures were linked to the severe degrees of sepsis which were associated with higher serum NGAL levels. Significant difference in serum NGAL level was found between survivors and non survivors, while no significant difference was found regarding PRISM III score.

Conclusion: Serum NGAL is significantly elevated in severe sepsis. Serum NGAL level was superior on the frequently used PRISM III score to predict mortality in critically ill patients with sepsis. So, serum NGAL can be used as a marker for severity and prognosis for mortality in septic pediatric patients.

Sepsis

Serum neutrophil gelatinase associated lipocalin

sepsis

pediatric intensive care unit.

Sepsis is a serious clinical illness that affects the body's physiology and biochemistry.[1] Severe sepsis is defined as that which results in organ dysfunction.[2] Septic shock is a severe form of sepsis exacerbated by acute circulatory failure that is marked by prolonged arterial hypotension in spite of adequate volume resuscitation and is not due to any other conditions.[3]

Each year, sepsis affects about 49 million individuals, and it's thought to be responsible for over eleven million deaths worldwide, or up to 19.7% of all fatalities.[4]. Overall mortality rates appear to be decreasing, although sepsis still loses the lives of up to 25% of patients. The hospital fatality rate for septic shock is close to 60%.[5]. 80 percent of deaths in children are categorized as sepsis-related deaths.[6] Decreasing mortality depends heavily on prompt identification, accurate classification, and care during the early stages of sepsis. [7]. Despite the fact that clinical scoring systems are valuable tools for assessing the severity and outlook of sepsis, the majority of them take 12 to 24 hours to complete, discovering biomarkers for early detection and assessing the prognosis of sepsis has enormous significance and takes a lot of time.[8] It has been noted that the existence of infection raises the levels of neutrophil gelatinase-associated lipocalin (NGAL).[9] NGAL is a 25-kDa protein that belongs to the lipocalin class.[10] NGAL expression was initially discovered in neutrophils and has been suggested that it exerts bacteriostatic effects, but its production has also been noted in the epithelia in reaction to oxidative stress. [11] Numerous findings link plasma-NGAL to immunological response, and a significant association involving plasma-NGAL, the production of interleukin 6 (IL-6), IL-10, as well as tumor necrosis factor-alpha was discovered, raising the possibility that plasma-NGAL is connected to inflammation. [12] According to some research, people with septic conditions had higher serum levels of NGAL[12, 13]

The purpose of this study was to compare and correlate the serum NGAL value estimated at admission with the clinical course and severity of illness in critically ill children, and to determine if it could be used as a prognostic marker for sepsis in sick children admitted to the PICU. It was also compared and correlated with the PRISM III score, which is the most frequently employed predictive mortality score for children with serious illnesses admitted to the PICU.

Study design:

This prospective case-control study was performed in Minia University children hospital which is a tertiary teaching health care center in Al Minya, Egypt. This study was conducted according to the principles of Helsinki. The Minia faculty of medicine ethical committee had given its approval for this research, Approval No; 388:2022, date:19 September 2022. Informed consent was obtained from all subjects and/or their legal guardian(s).

Patient selection and eligibility criteria:

The study had conducted on 75 patients with sepsis encountered from the pediatric intensive care unit (PICU) and 25 apparently healthy age and sex matches children were enrolled as control group from September 2022 to March 2023. Patients were divided into 2 groups: Group Ι: Included 75 children with sepsis. Their ages ranged was 1–12 years. Patients in this group were subdivided into Sepsis subgroup = 25 Cases, Septic shock subgroup = 25 Cases and Multiple organ dysfunction subgroup = 25 Cases.

GroupΙΙ

Included 25 apparently healthy age and sex matched children as controls.

Inclusion criteria: all pediatric critical care unit admissions under the age of 12 who met the criteria for septicemia. Exclusion criteria: 1) newborn, those over than 12 years, those with no blood test taken within one hour of arrival into PICU, and the absence of parental permission.

According to The American College of Chest Physicians/Society of Critical Care Medicine criteria for sepsis, which were updated specifically for pediatrics, were used for the diagnosis our patients. Septicemia is considered by the following criteria were used: 1) Core temperature of greater than or less than 38.5°C (Temperature measurement was done using the axillary method), 2) increased heart rate, 3) increased respiratory rate, 4) high or low total leukocyte count for age, or 10% immature neutrophils, and 5) A positive blood culture along with the signs and symptoms of the main illness.

When any of the following clinical indicators of insufficient tissue perfusion were present, septic shock was determined to be present: 1)Reduced or changed mental status, 2) prolonged refills of capillaries lasting longer than 2 seconds, 3) weaker pulses, 4) mottled chilly extremities, 5) reduced urine production of less than 1 ml/kg / hour, and 6) continued hypotension in spite of a sufficient supply of fluids. Along with the signs and symptoms of septicemia. [14]

Data measurements:

Data were collected on patient’s background characteristics and his laboratory investigations:

Thorough history-taking and physical examination includes a general examination, abdominal examination, neurologic assessment, and evaluation of the chest and heart.

Laboratory investigations: complete blood count, random blood glucose. Tests of Renal Function. (creatinine and urea). ALT, AST, TB, and DB Time and concentration of prothrombin (PT, PC) and PTT. The blood cultures C reactive protein (CRP), serum electrolytes and arterial blood gases

PRISM score type III: [15]

The Following parameters were taken from previous data of clinical examination and lab investigations and were combined to perform PRISM-III system for scoring: Heart and vascular system tests include mean arterial blood pressure and pulse. Nervous system tests include pupils' light reflex and the state of consciousness. Blood tests include arterial blood gases, blood glucose level, renal function tests, platelets and white cell numbers, PT, and PTT, core temperature. Information was inputted into a PRISM III calculator that was made accessible electronically at www.medal.org.[16] The PRISM III scores are interpreted as follows: 0–5, 6–10, 11–15, 16–20, 21–25, and 26–30. With probability of death is 11%, 23%, 40%, 61%, 78% and 89% respectively.

Specific investigations: All patients were measured for NGAL once within the first hour of their admission to the PICU, along with the control group. An approved commercial dual monoclonal antibody (MA) sandwich immunoassay kit (Human Neutrophil Gelatinase-Associated Lipocalin ELISA Kit) was used to measure the levels of serum NGAL.; Glory Bioscience, Del Rio, TX).[17]

Statistical analysis:

SPSS version 22 (Statistical Software package version 22) was used to analyze the data. There was a descriptive analysis done. Mean, SD, and range were used to depict quantitative data. The Chi-square comparison test for qualitative results across groups was used to compare frequency and percentage reports of qualitative data. Graphs were created using SPSS version 22 or Excel. P-value was less than 0.05 were considered significant. For non-parametric quantitative information between the four groups, the Kruskal Wallis test is used, subsequently followed by the Mann Whitney test for each pair of groups. A one-way ANOVA test for quantitative parametric variables comparing the four groups, followed by post-hoc Bonferroni test for each pair of groups.

This study was carried out on 75 patients with sepsis enrolled from the pediatric intensive care unit and 25 apparently healthy children controls. Our study found that there were significant differences among studied groups regarding laboratory data; the mean of serum NGAL was highest among the multiorgan failure group and was lowest among the control group (Table 1 and Fig. 1). On the same hand, the multiorgan failure group showed significantly highest means regards CRP, TLC, and RBS (Table 1). Also, we found the mean of PC was highest among the sepsis group, and INR and PTT were highest among the Multiorgan failure group. (Table 2). On performing correlation between NGAL and other clinical and laboratory parameters, there were significant fair negative correlations between age, Ca, and Hb with NGAL, but there were strong positive correlations between CRP, TLC, urea, TB, INR, and PTT with NGAL (Table 3). However, on performing linear regression analysis to show predictors of elevated serum NGAL, only CRP, INR, and PTT were found to be significant predictors (Table 4). Regarding PRISM III scores among different studied groups. The Mean PRISM III score was (30.32 ± 4.09) in the Multiorgan failure group versus (24.96 ± 2.81) in the severe sepsis group and (16.04 ± 3.43) in the sepsis group. The differences were statistically significant (Table 5). While in measuring and comparing serum NGAL levels and PRISM III scores between survivors and non survivors, significant difference was observed between the 2 groups only in serum NGAL levels but not for PRISM III scores rendering serum NGAL levels have superior prognostic value over PRISM III scores (Table 6). Severe sepsis group and multiorgan failure group had positive cultures while (28%) of specimens in the sepsis group had no bacterial growth, referring that bacterial infection associated with more sever degrees of sepsis which were associated with higher serum NGAL levels. Half of the positive cultures in the severe sepsis group and multiorgan failure group showed klebsiella, and the second most prevalent organism was E-coli followed by staphylococcus aureus. (Fig. 2).

In this study, the serum NGAL was significantly increased in critically ill children with severe grades of sepsis (severe sepsis and septic shock) compared to healthy ones. Also, serum NGAL in critically ill children with septic shock was significantly higher than in children with severe sepsis, reflecting increased serum level with the severity of illness. A significant difference in serum NGAL level was found between survivors and non survivors in patients group, which indicate that the mortality rate was linked to highest serum NGAL levels.[18, 19] Furthermore, the non survivors had greater median and interquartile range and increased serum level of NGAL levels than the admitted PICU survivors did.[20, 21]

The multiorgan failure patients showed significantly the highest means regarding CRP, PC, INR and PTT. The previously mentioned laboratory markers are effective discriminators of sepsis severity indicating worse prognosis in such patients.[22–24]

On trying to correlate routine laboratory parameters as blood elements, coagulation profile, liver function tests, renal function tests, random blood sugar and calcium levels with the measured serum NGAL, all the previously mentioned laboratory parameters showed direct positive correlation with serum NGAL, however when performing linear regression analysis to show predictors of elevated serum NGAL only CRP, INR, and PTT were significant predictors for serum NGAL. This matched the results of Cai et al., 2022 confirming that in the diagnosis of newborn sepsis, the amount of sNGAL was substantially associated with the level of CRP and PCT.[25]

The PRISM score is a metric that is widely used in PICUs to forecast patient death. Its great specificity and sensitivity for estimating the death rate of admitted patients are demonstrated in numerous investigations.[26, 27]

Significant differences were found between mean PRISM scores in different sepsis groups with highest score in the multiorgan failure, while in comparing the score between survivors and non survivors no significant differences had been found. The poor PRISM score III prediction compared to reported mortality was previously performed and worse worse outcome was proved in individuals with respiratory illnesses. Among the PICU sepsis patients, a higher serum NGAL level was evaluated during the first hour of admission is superior to other methods of predicting the severity and survival rate.[28]

According to blood cultures, about (28%) of specimens in the sepsis group had no bacterial growth and almost always severe sepsis group and multiorgan failure group had positive cultures, suggesting that higher levels of serum NGAL can indicate that severe degrees of sepsis is mainly related to bacterial infections.[29] Individuals with bacterial infections had much greater levels of NGAL than the healthy group did. [30, 31] and hepatic patients with bacterial infections had greater serum levels of lipocalin-2 than individuals without bacterial infections.[32] A previous study in Egypt revealed that patients with infection had significantly higher serum levels of NGAL in a research on Egyptian children who had persistent liver disease. [33]

In our study, most of the PICU admitted patients diagnosed as pneumonia followed by gastroenteritis and then CNS infections. Pneumonia contracted from the community, gastroenteritis, and meningitis were the most frequently identified infections necessitating PICU hospitalization.[34]

In the study of Min et al., 2020, 241 hospitalized patients with pneumonia, examined the relationship between plasma levels of NGAL, admission to the intensive care unit, and in-hospital death. Patients who were hospitalized to the ICU had NGAL levels that were noticeably greater than the ones who were discharged. Similar to this, patients who did not survive had a higher likelihood of having higher NGAL levels than those who did. [35–37]

Previous reports for patients with bacterial meningitis, high levels of NGAL in their CSF were found.[38, 39] Additionally, NGAL is a highly sensitive and reliable marker for differentiating bacterial meningitis from a variety of other CNS illnesses.[40]

Nearly half of the culture (52.6%) from the multi-organ failure group and around (47%) from the severe sepsis group had Klebsiella growth. Klebsiella pneumoniae is among the most prevalent bacteria and the sepsis caused by Klebsiella pneumonia can considerably raise the likelihood of systemic multi-organ failure. [41] We also observed that around 25% of the results of positive cultures are due to E. coli growth, and 15.6% are due to staphylococcus aureus growth.

Numerous researches looked into the increased serum NGAL levels in various bacterial infections. Bacteria produce siderophores during processes of inflammation. Because of siderophores' high affinity towards iron, lactoferrin and transferrin are broken down and the iron is transferred into the organism that causes the illness [42] Toll-like receptor (TLR) activation of the macrophage activates the NGAL gene and increases the production of NGAL. NGAL expression is induced by pro-inflammatory cytokines in neutrophils, cells of the epithelium, or liver cells.). [43, 44] Because the ability of the pathogen to multiply depends frequently on the bioavailability of iron, NGAL sequesters siderophores, stops bacteria from getting iron, and hence reduces bacteria growth as well as replication. (Parrow et al., 2013). [42]

In particular, it has been seen that NGAL inhibits Escherichia coli's formation of siderophores, which has been involved in pneumonia. Additionally, the respiratory tract infection with Escherichia coli increases NGAL production in airways epithelium as well as alveolar type II pneumocytes.[45]

NGAL defends the respiratory system from infections caused by Klebsiella pneumoniae, Staph aureus, or M. tuberculosis. [46–48] Although NGAL's primary bacteriostatic action is the sequestration of pathogenic bacterial siderophores, it also plays a role in the stimulation and differentiation of T-cells into the Th1 subtype. (Floderer et al., 2014). [49]

The serum NGAL level can be a good prognostic marker for sepsis severity especially in bacterial sepsis and can predict the disease outcome for patients admitted in pediatric intensive care units. It can be a good predictor for the risk of sepsis-related death.

Limitations:

The high cost of the s NGAL limited the researchers to screen it on a wide scale of populations.

ALT: Alanine aminotransferase

AST: Aspartate aminotransferase

Ca: Calcium

CBC: Complete blood count

CNS: Central nervous system

CRP: C reactive protein

DB: Direct bilirubin

ESR: Erythrocytic sedimentation rate

HB : Hemoglobin

MA: monoclonal antibodies

PICU: Pediatric intensive care unit

PT: Prothrombin time

PTT: Partial thromboplastin time

RBCs: Red blood cells

sNGAL: Serum neutrophil gelatinase associated lipocalin

TB: Total bilirubin

TLC: Total leucocytic count

TLR: Toll-like receptor

The study was approved by the local ethics committee of the Minia University faculty of medicine (IRB 88:2022) at 19 September 2022.

Consent for publication:

Not applicable

Availability of data and materials:

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests:

The authors declared no conflict of interest.

Funding:

The research is funded by the authors own, no institutional funding

Authors' contributions:

All authors shared the selection of the research subject. GL, ME and ES made the study design. MS collect the patient data, OM made the manuscript drafting, ME and ES made the data analysis and interpretation. MS, ME, ES and OM shared research drafting and revision, GL revised and edited the final manuscript. All authors approved the final draft.

Acknowledgements:

Non

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Table (1) Differences between studied groups according to laboratory data

Variables	Sepsis N=25	Severe sepsis N=25	Multiorgan Failure N=25	Control N=25	Significance
s.NGAL	532.56±26.59	671.8±49.71	1534.12±467.47	365.4 ±60.50	p=0.001*
					p1=0.05*		p2=0.001*	p3=0.03*
					p4=0.001*		p5=0.001*	p6=0.001*
CRP	12.72±6.99	27.84±14.99	66.24±27.95		p=0.001*
					p1=0.006*	p2=0.001*
					p4=0.001*
Hb	9.82±0.52	8.56±0.55	8.4±0.94	11.55 ±0.91	p=0.001*
					p1=0.001*	p2=0.001*			p3=0.001*
					p4=0.47	p5=0.001			p6=0.001
TLC	12.71±1.31	14.8±1.25	23.97±6.67	8.02 ±1.16	p=0.001*
					p1=0.04	p2=0.001*			p3=0.001*
					p4=0.001*	p5=0.001*			p6=0.001*
PLT	158.56±7.53	138.36±8.76	91.48±35.49	311.2 ±51.97	p=0.001*
					p1=0.02*	p2=0.001*			p3=0.001*
					p4=0.001*	p5=0.001*			p6=0.001*
RBS	171.56±17.76	268.52±20.47	279.04±173.84		p=0.001*
					p1=0.001*	p2=0.001*
					p4=0.72

Analyzed by Mann-Whitney Test, *: Significant difference at P value < 0.0P1: between sepsis and severe sepsis groups, P2: sepsis and Multiorgan failure, P3=Sepsis and control, P4: severe sepsis and Multiorgan failure, P5: severe sepsis and control, P6: Multiorgan failure and control

Table (2) Coagulation tests among different studied groups

Variables	Sepsis N=25	Severe sepsis N=25	Multiorgan Failure N=25	Significance
PC	80.72±2.92	70.32±5.27	41.96±9.78	p=0.001*
				p1=0.001*	p2=0.001*	p3=0.001*
INR	1.02±0.08	1.34±0.16	1.56±0.55	p=0.001*
				p1=0.001*	p2=0.001*	p3=0.03*
PTT	29.84±3.02	39.12±1.74	45.96±4.9	p=0.001*
				p1=0.001*	p2=0.001*	p3=0.001*

Analyzed by Mann-Whitney Test, *: Significant difference at P value < 0.05

P1: Sepsis &severe sepsis, P2: Sepsis & Multiorgan failure, P3: severe sepsis, Multiorgan failure

Table (3) Correlation between NGAL and other parameters

Table (4) Linear regression analysis to show predictors of elevated serum NGAL

Variable	Standardized coefficient B	Confidence interval	Significance
Septic group	0.104-	(-312.47- -180.99-)	0.59
Gender	0.003-	(-87.72-81.83)	0.95
Age	0.004	(-18.28-19.68)	0.94
CRP	0.18	(0.25-6.28)	0.03*
Hb	0.09	(-23.66-121.31)	0.18
TLC	0.08	(-7.52-21.27)	0.34
PLT	0.14-	(-5.99-1.98)	0.32
RBS	0.06-	(-0.77-0.22)	0.27
PRISM	0.07-	(-20.41-9.32)	0.46
ALT	0.34-	(-1.56-0.73)	0.47
AST	0.46	(-0.86-2.15)	0.39
TB	0.29	(-30.61-381.15)	0.09
DB	0.14-	(-594.96-191.37)	0.31
Urea	0.19-	(-9.26-0.52)	0.08
Creatinine	0.16-	(-206.14-34.64)	0.16
Na	0.09-	(-25.23-3.96)	0.15
K	0.03-	(-208.69-157.86)	0.78
Ca	0.07-	(-791.41-118.84)	0.14
PC	0.29-	(-17.38-0.25)	0.06
INR	0.35	(218.06-693.51)	0.001*
PTT	0.33	(6.6-39.01)	0.007*

*: Significant difference at P value < 0.05

Table (5) PRISM score among different studied groups

PRISM score

Sepsis

Severe sepsis

Multiorgan failure

Significance

Mean ± SD

Range

16.04±3.43

(10-21)

24.96±2.81

(20-30)

30.32±4.09

(22-39)

P=0.001

P1=0.001

P2=0.001

P3=0.001

Analyzed by Mann-Whitney Test, *: Significant difference at P value < 0.05P1: between sepsis and severe sepsis groups, P2: sepsis and Multiorgan failure, P3= severe sepsis and Multiorgan failure.

Table (6) Association between s. NGAL, PRISM scores and patient outcome

Survived

N=21

Died

N=54

Significance

S. NGAL

(mean ± SD)

642.52 ± 148.1

1017.94 ± 574.1

P=0.0001*

Prism score

(mean ± SD)

21.33 ± 6.9

24.72± 6.65

P=0.05

Analyzed by Mann-Whitney Test, *: Significant difference at P value < 0.05

No competing interests reported.

Impact of Neutrophil Gelatinase-associated Lipocalin A Novel Sepsis Biomarker on PRISM III Score in Predicting Sepsis Severity And Mortality in Pediatric Intensive Care Units (Single Center Study)

Status:

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Abstract

Figures

Introduction

Patients and Methods

Study design:

Patient selection and eligibility criteria:

Data measurements:

Statistical analysis:

Results

Discussion

Conclusion

Limitations:

Abbreviations

Declarations

References

Tables

Additional Declarations

Status:

Version 1