Role of Serum Apelin in the Diagnosis of Early-Onset Neonatal Sepsis.

OBJECTIVE: Apelin is a proinflammatory adipocyte-derived factor. The aim of this study was to evaluate the role and significance of serum apelin as a new sepsis marker in the identification of full-term and preterm new-born infants with early-onset sepsis.
MATERIAL AND METHODS: This was a case-control study. We included 80 neonates. The cases were divided into 2 groups; neonates with early-onset sepsis and control group with neonates non-sepsis. Apelin was quantified by enzyme-linked immunosorbent assay.
RESULTS: There was a significant elevation in the mean values of serum apelin in the early-onset sepsis group (1214.7 ± 273.06 pg/mmol) than in the non-septic neonates 116.27 ± 21.96 pg/mmol (P < .0001). Apelin values were correlated to clinical sepsis and hematological scores as well as C-reactive protein. Serum apelin concentration was significantly higher among culturepositive cases than the culture-negative cases (mean ± SD was 1239.52 ± 268.47 and 929.42 ± 136.97 pg/mmol, respectively, P < .0001). Moreover, the apelin level was higher in non-survivor neonates than in the survivors in the early-onset sepsis group. No significant difference was found between preterm and full-term new-born infants with regard to the apelin values. The best cut-off estimate of apelin to diagnose early sepsis was >178.33 pg/mmol.
CONCLUSION: Apelin may be useful in the diagnosis and prognostic prediction of neonates with early-onset sepsis.

Neonatal infection is an important cause of mortality. Prompt management of the new-born infants with suspected sepsis may improve the survival of neonates.

The primary diagnosis of neonatal infection is still a challenge for neonatologists due to the lack of highly sensitive and specific markers with accepted cut-off values.

Apelin is a proinflammatory factor that contributes to inflammation.

Apelin has a significant reliable diagnostic and prognostic value for detecting early sepsis in new-born infants.

Serum apelin can be used in the prediction of neonates with early-onset sepsis (EOS).

The apelin value can predict EOS in full-term and preterm new-born infants.

Introduction

Neonatal infection is one of the major causes of morbidity and mortality among neonates. Each year, about one million new-borns in developing countries die of infection, with a risk of neurological damage seen in survivors.[1]

A positive blood culture is considered the “gold standard” to diagnose neonatal sepsis,[2] but it provides an inadequate sensitivity to start antibiotic therapy.[3] When blood and other sterile site cultures are negative but the infant manifests a sign consistent with infection, they may be considered to have “clinical” sepsis.[4,5]

The initial treatment is to regularly use antibiotics when sepsis is suspected, but the diagnosis is delayed due to suspicion and then diagnosed with increasing danger. Prompt, precise, and fast identification of neonatal sepsis still an analytical dispute, highlighting the demand for consistent and appropriate analytic markers to provide effective antibiotic management.

Apelin is the endogenous ligand for the G-protein-coupled APJ receptor. This is expressed at the surface of some cell types. It is widely expressed in various organs such as the heart, lungs, kidney, liver, adipose tissue, gastrointestinal tract, brain, adrenal glands, endothelium, and human plasma. Both mouse and human adipocytes express and secrete apelin, which is a proinflammatory adipocyte-derived factor that participates in vascular wall inflammation.[6,7] Apelin has been reported to mediate a variety of physiological actions.[8] It showed alteration in adults with severe sepsis. However, there is evidence for its diagnostic and prognostic benefits.[9] Apelin amends heart damage in sepsis by tempering inflammatory reactions and could hopefully be a target to manage severe sepsis.[10]

Research question: Can we use apelin as an early indicator for the identification of EOS in new-born infants?

The aim of this study was to evaluate the role and significance of serum apelin as a new sepsis marker in identifying the full-term and preterm new-born infants with EOS.

Methods

Materials

Type of Study

This case-control study included 80 new-born infants. The sample size was determined by the electronic calculator from https://epitools.ausvet.com.au/casecontrolssequation. The confidence level was 0.95, power was 0.8, and assumed odds ratio was 4, for the data used. Accordingly, we determined a minimum of 39 cases for each group.

Study Groups

The neonates in the study were assigned into 2 groups:

Group I: Early-onset sepsis (EOS). It included 50 neonates [25 full-term neonates and 25 preterm infants (32-34 weeks)] with early-onset neonatal sepsis, diagnosed by clinical examination and laboratory investigation.

Group II: Non-sepsis group. It included 30 neonates (15 full-term neonates and 15 preterm infants). They were healthy new-born infants with no signs of infection, negative C-reactive protein (CRP), and non-suggestive hematological results.

Inclusion Criteria

The inclusion criteria for the neonates with sepsis included the presence of at least 2 clinical symptoms and at least 2 laboratory signs in the presence of, or as a result of, suspected or proven infection (positive culture or microscopy polymerase chain reaction) within the first 72 hours.[11]

Procedures

The full history, clinical examination, Töllner clinical sepsis score,[12] and serum apelin levels were obtained for all the studied cases. Routine laboratory investigations including complete blood count with differential count, hematological score,[13] blood culture, and CRP were also obtained.

The diagnosis of sepsis in the new-born infant was based on the presence of at least 2 clinical symptoms such as temperature instability, cardiovascular and/or respiratory instability including bradycardia or tachycardia, hypotension, apnea, tachypnea or increased oxygen requirements, feeding intolerance, poor suckling, abdominal distension, irritability, lethargy, and hypotonia, in addition to at least 2 laboratory findings including complete blood count, positive CRP, thrombocytopenia, metabolic acidosis, and positive culture. Abnormal radiological findings of pneumonia and necrotizing enterocolitis were also considered. A case with a Töllner score > 3 and hematological score > 5 was diagnosed as sepsis. Measurement of serum apelin was achieved using enzyme-linked immunosorbent assay (MyBioSource, Inc.; catalog number MBS756419_competitive). The research was approved by the Ethics Committee of the Faculty of Medicine for girls, AL-Azhar University. The approval number is 202103752.

Statistical Analysis

Data were collected, coded, revised and entered into the Statistical Package for the Social Sciences (SPSS) version 25.0 (IBM SPSS Corp.; Armonk, NY, USA). The data were presented as numbers and percentages for qualitative data, mean and standard deviations (SD) and ranges for the quantitative data with parametric distribution, and median with interquartile range (IQR) for the quantitative data with non-parametric distribution.

Student’s t-test was used in the comparison between the mean of 2 groups with regard to quantitative data and parametric distribution. For non-parametric data, the Mann–Whitney U-test was used to compare the nonparametric results that were not normally distributed.

Pearson’s linear correlation coefficient (r) was estimated in order to show the relationship between quantitative parameters.

The receiver-operating characteristic curve was used to assess the best cut-off point between the 2 groups with its sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under curve (AUC). The AUC was interpreted as follows; 0.9-1, excellent biomarker; 0.8-0.9, good; 0.70-0.80, fair; and 0.60-0.70, poor.

The confidence interval was set to 95% and the margin of error accepted was set to 5%. Therefore, the P-value was considered significant at < .05.

Results

The results are shown in Tables 1, 2, 3, 4 and 5. Table 1 shows the distribution of demographic data among the studied groups, while Tables 2-5 show the laboratory findings.

Table 1.

Distribution of Demographic Data According to Patient Groups

	Sepsis Group	Non-sepsis Group	Significant Test	P
Gestational age (weeks)
Range	32-39	32-39	0.627*	.532
Mean ± SD	35.32 ± 2.71	35.7 ± 2.48
Postnatal Age (hours)
Range	25-64	27-62	0.843*	.180
Mean ± SD	42.88 ± 11.9	35.73 ± 12.5
Gender
Male, N (%)	23 (46.0)	13 (43.3)	0.054^	.816
Female, N (%)	27 (54.0)	17 (56.7)
Birth weight (kg)
Range	1.00-5.200	1.330-4.200	2.73*	.257
Mean ± SD	2.370 ± 1.00	2.656 ± 0.729
Length (cm)
Range	30-54	37-54	1.575*	.119
Mean ± SD	44.06 ± 5.85	46.02 ± 4.51
Apgar 1 min
Median	5	8	108.000#	.000
IR (25%-75%)	5-6.25	7-.8
Apgar 5 min
Median	8	10	98.500#	.000
IR (25%-75%)	8-9	10-10
Resuscitation
Needed, N (%)	41 (82)	0 (0)	50.462^	.000
Not needed, N (%)	9 (18)	30 (100)
PROM > 18 h
Yes, N (%)	27 (54.0)	5 (16.7)	10.889^	.001
No, N (%)	23 (46.0)	25 (83.3)
PROM covered by antibiotics
Yes, N (%)	23 (46.0)	21 (70.0)	6.299^	.043 S
No, N (%)	5 (10.0)	4 (13.3)
Blood transfusion
Yes, N	10	0	6.857^	.009
No, %	20.0	0.0
Survival
Live, (%)	32 (64.0)	30 (100)	13.935^	.000
Dead, N (%)	18 (36.0)	0 (0)

N, number; PROM, premature rupture of membrane; * t-test; ^chi-square test; #Mann–Whitney rank-sum test.

Table 2.

Comparison Between the Studied Groups Regarding Serum Apelin and C-Reactive Protein

Apelin	Serum Apelin, pg/mmol							Independent t-test
Mean	SD	Minimum	Maximum	Median	IR (25%-75%)	Range	t	P
Sepsis group	1214.7	273.06	683.33	2112.33				28.29	.000
Non-sepsis group	116.27	21.96	78.5	178.33
Gender
Male	1167.6	339.12	683.33	2112.33				−1.128	.265
Female	1254.8	198.96	690.66	1556.67
Sepsis Group
Full-term	1153.6	245.66	683.33	1550.67				−1.606	.115
Preterm	1275.7	290.03	815.33	2112.33
Non-sepsis group
Full-term	110.17	12.41	93.33	132.5				−1.560	.130
Preterm	122.38	27.66	87.5	178.33
Preterm
Non-sepsis	122.38	27.66	87.5	178.33				15.281	<.001
Sepsis	1275.7	290.03	815.33	2112.33
Full-term
Non-sepsis	110.17	12.41	93.33	132.5				16.353	<.001
Sepsis	1153.6	245.66	683.33	1550.67
Blood culture
Positive blood culture	1239.5	268.47	683.33	2112.33				2.269	.028
No growth	929.42	136.97	817.33	1112
Outcome
Live	1112.1	242.78	683.33	1779.17				−4.062	.001
Dead	1397.0	229.03	901.33	2112.33
Postnatal age
2nd day	510.06	502.04	87.50	2112.33				−2.506	.015
3rd day	848.88	522.77	93.33	1556.67
CRP mg/dl								109.5	.000
Sepsis					24	24-48	2-96
Non-Sepsis					4.5	3-6	2-6

CRP, C-reactive protein.

Table 3.

Cut-Off Point, Sensitivity, and Specificity of Serum Apelin, CRP, Töllner, and Hematological Score.

Test	AUC	Cut-Off Value	Sensitivity	Specificity	PPV	95% CI		NPV	95% CI
Lower bound	Upper bound	Lower bound	Upper bound
Apelin	1.000	>178.33	100.00	100.00	100.0	92.9	100.0	100.0	88.4	100.0
CRP	0.950	>6	90.00	100.00	100.0	92.1	100.0	85.7	69.7	95.2
Töllner score	0.961	>5	82.00	100.00	100.0	91.4	100.0	76.9	60.7	88.9
Hematological score	0.939	>2	74.00	100.00	100.0	90.5	100.0	69.8	53.9	82.8

This table shows that at the cut-off point of >178.33 pg/mmol serum apelin has 100% sensitivity, 100% specificity, negative predictive value (NPV) 100%, and positive predictive value (PPV) 100% in the diagnosis of EOS. The cut-off point of > 6 mg/dl C-reactive protein has 90% sensitivity, 100% specificity, NPV 100%, and PPV 85.7% in the diagnosis of EOS. The cut-off point of > 5 Töllner score has 82% sensitivity, 100% specificity, NPV100%, and PPV 76.9% in diagnosis of EOS. The cut-off point of >2 hematological score has 74 % sensitivity, 100% specificity, NPV 100%, and PPV 69.8% in the diagnosis of EOS.

Table 4.

Cut-Off Point, Sensitivity, and Specificity of Serum Apelin in Preterm and Full-Term Infants

GA	Cut-Off Point	AUC	Sensitivity	Specificity	NPV	PPV
Preterm	>178.33	1.000	100.00	100.00	100.0	100.0
Full-term	>132.5	1.000	100.00	100.00	100.0	100.0

This table shows that at the cut-off point of >178.33 serum apelin has 100% sensitivity, specificity, negative predictive value, and positive predictive value for diagnosis of EOS in preterm neonates. The cut-off point of >132.5 has 100% sensitivity, specificity, negative predictive value and positive predictive value in the diagnosis of EOS in full-term neonates.

Table 5.

Correlation Between Serum Apelin and Different Parameters in the Sepsis Group

	Serum Apelin
r	P
Gestational age	−0.209	.145
Postnatal age	−0.054	.708
Gestational age	−0.209	.145
Duration of premature rupture of membrane	0.410	.003
Birth weight	−0.316	.026
Mean blood pressure	−0.377	.007
C-reactive protein	0.701	.000
Töllner score	0.807	.001
Hematological score	0.48	.001
Hospital stay	0.087	.549

Description of the study population

The study group included 50 neonates with EOS (27 females [54.0%] and 23 males [46. 0%]).The mean age of the participants was 42.88 ± 11.9 hours.

There were no statistically significant differences in gestational age, birth weight, postnatal age, and gender between the sepsis and non-sepsis group. The Apgar score at 1 and 5 minutes decreased in the sepsis group compared to the non-sepsis group.

The Töllner score was significantly elevated in the sepsis group (P < .001).

Clinical Presentation

Respiratory distress was the most common clinical presentation in the EOS group (30%), followed by hypothermia (28.3%), and the least common clinical presentation among the cases studied was seizure (5%). Hypothermia was significantly increased among preterm infants.

Risk Factors

The number of cases with PROM (>18 hours) was significantly higher in the sepsis than in the non-sepsis groups.

Laboratory Findings

Forty-six newborns (92.0%) were proved to have sepsis by positive blood cultures. The bacteria identified as included Gram negative in 71.7% of the cases and Gram positive in 28.3%.

Blood culture was negative in all the cases of the control group. The hematological score was elevated among infants with EOS. The sepsis group presented with significant increase in total leukocyte, neutrophil counts, immature neutrophil, immature to mature, and immature to total neutrophil ratios.

Discussion

This study illustrated that there was an increase in the level of serum apelin in the sepsis group compared to the non-sepsis group. Gad et al.[14] found an 8-fold increase of serum apelin in neonates with sepsis compared to the controls. Apelin is upregulated following sepsis, and plays a protective role in sepsis-induced cardiac impairment.[15] It is possible that the upregulation of apelin in response to inflammation is a compensatory process to regulate the onset of metabolic disturbances. The release of apelin is controlled by inflammatory mediators, such as TNF-α, IL-6, and IFN-γ.[16]

In this study, gender has no effect on apelin values in the sepsis and in the non-sepsis groups. This finding is similar to Malamitsi-Puchner et al.[17] Moreover, gestational age had no effect on serum apelin values, as there was non-significant difference between the full-term and preterm neonates in the sepsis group and in the non-sepsis cases.

Although the Apgar score was lower in the EOS group at the first and fifth minutes than in the non-sepsis group, it was within normal range at the fifth minute. This finding excluded perinatal asphyxia. A lower Apgar score in new-borns with EOS is reported by others.[18,19] Some isoforms of apelin, such as APLN-13 and APLN-36 inhibit ROS, and improve the oxidative stress.

In this study, serum apelin was significantly higher in the positive blood culture cases than in those with negative blood culture. The percentage of positive blood culture in the sepsis group was 92%. Gram-negative organisms were detected in 71.7% of these cases. This was slightly higher than the results of Boraey et al.,[20] who reported positive blood cultures in 81.2% of the cases. On the other hand, the study of Arif et al.[21] showed that only 15% of septic cases had positive blood cultures and concluded that more than half of the cases of neonatal sepsis are missed if only blood culture is used as the basis of diagnosis. This variation may be due to the differences in the etiology and stage of sepsis as well as start of antibiotic therapy prior to the laboratory diagnosis, which may be the major cause for the low culture results.

Apelin was significantly elevated in the non-survivor new-born infants than in the survivors. Severe infection in very ill cases led to the release of more apelin. In elderly patients with sepsis, Wang et al.[22] reported a lower level of apelin in the survival group than in the mortality group.[22] Body fluid homeostasis is among the physiological functions of the apelinergic system, which can be useful in the treatment of the sepsis.[23]

APLN-13 significantly increases survival and improves left ventricular performance, better than the continuous infusion of dobutamine. It also reduces inflammation and stress.[24]

The best value for apelin for diagnosis of EOS was >178.33 pg/mmol. This value has high sensitivity and specificity. Comparative results were reported by Gad et al.[14] but with a lower cut-off value.[14] The variations in cut-off values can be attributed to different laboratory methods, sampling techniques (plasma, serum, or whole blood), and the characteristics of the studied populations such as postnatal age, birth weight, proportion of positive culture cases, and severity of illness.

The cut-off value of apelin was higher in immature cases, which may be related to modulation of the apelinergic system throughout pregnancy. Apelin expression levels had slightly decreased from the first to the third trimester of gestation due to the accelerated placental metabolism.[25]

The Töllner score had an AUC of 0.961 with a cut-off value > 5 with 82% sensitivity and 100% specificity, NPV 76.9%, and PPV 100%, for diagnosis of EOS in new-born infants. In addition, the hematological sepsis score had an AUC of 0.939 with a cut-off value > 2, with 74.00% sensitivity and 100% specificity, 69.8% NPV, and 100% PPV. Therefore, apelin had the highest AUC, sensitivity, specificity, NPV, and PPV, which makes the serum apelin level the most accurate parameter in the prediction of EOS. The Töllner and hematological sepsis scores can be used as initial diagnostic tools to predict EOS in the neonatal population, especially in developing countries.[26]

A negative association was noticed between serum apelin and mean blood pressure. An experimental apelin injection lowered the blood pressure of study animals.[27] Apelin has vasodilator effects attributed to nitric oxide.[9]

In the present study, a non-significant positive association was detected between serum apelin and length of hospital stay. Cömert et al.[28] reported a significant positive correlation between the presence of sepsis and prolonged NICU stay.

Finally, this study showed that the serum apelin may be a useful and novel biomarker, as it is able to distinguish septic patients from other patients admitted to the NICU and is also able to predict sepsis severity and outcome. Additional information and studies for exploring the new family of apelinergic drugs to replace catecholamines in the treatment of sepsis are required.[29]

The application of safety standards for the neonatal patient, as well as the strict dedication to measures such as hand hygiene and the proper usage of antibiotics are considered mandatory to reduce infections in the NICU.[30]

The limitations of This Study

We could not determine how long it takes for apelin levels to rise after infection. It may need other experimental studies.

We have reached the conclusion that a negative blood culture does not exclude sepsis. Apelin was found to be of significant reliable diagnostic and prognostic value in the diagnosis of EOS among preterm and full-term infants. Serum apelin can be used in predicting neonates with EOS.

The Take-Home Message

Neonatal sepsis is still the major cause of mortality and morbidity in the NICU. Although sepsis markers such as apelin show promising early diagnostic value, adherence to infection control policies––including attention to strict hand hygiene, antibiotic stewardship, and catheter management––is required to decrease the number of infections in hospitalized neonates.