Pentraxin 3 as a novel diagnostic marker in neonatal sepsis

Abstract

BACKGROUND:

Neonatal sepsis is an important cause of morbidity and mortality especially in developing countries. As clinical manifestations of neonatal sepsis are nonspecific, early diagnosis and treatment remain a challenge. Pentraxin 3 (PTX3) is an acute-phase protein secreted by various cells in response to the proinflammatory signals. Our aim was to investigate the diagnostic value of PTX3 in neonatal sepsis.

METHODS:

We studied 90 neonates; 60 with culture-proven sepsis and 30 healthy neonates as a control group. Serum levels of PTX 3 were measured by ELISA.

RESULTS:

Neonates with sepsis had significantly higher levels of PTX 3 as compared to controls (p < 0.001). Diagnostic cutoff value of PTX 3 was 5.6 μg/L with a sensitivity of 98.3% and a specificity of 96.7%. PTX 3 was significantly increased in nonsurvivors when compared to survivors (p < 0.001). PTX3 had better sensitivity when compared with CRP.

CONCLUSION:

PTX 3 could be used as a new biomarker of neonatal sepsis with high sensitivity and specificity.

Keywords

Diagnosis marker neonate pentraxin 3 sepsis

1 Introduction

Every year about one million infants die worldwide because of neonatal sepsis and most of these deaths occur in low resource developing countries [1]. The impact of neonatal sepsis extend beyond mortality, to long-term complications in survivors. Early treatment of neonatal sepsis is associated with improved outcomes, so that rapid diagnosis is key to reducing this burden [2].

The most common risk factors associated with early onset sepsis (EOS) are preterm delivery, low birth weight, premature rupture of membranes, maternal urinary tract infection and maternal group B Streptococcal colonization while late onset sepsis (LOS) is associated with central/umbilical catheters, mechanical ventilation and parenteral nutrition [3].

Clinical manifestations of neonatal sepsis are nonspecific. These include temperature instability, respiratory distress, apnea, poor feeding, lethargy, irritability, convulsions, hypotonia, poor perfusion and abdominal distension. Blood culture is the gold standard for the diagnosis of sepsis; however, the false-negative rate is high and the result is frequently delayed [4, 5].

Pentraxins are evolutionarily conserved group of pattern recognition glycoproteins which share a pentraxin like domain in the C terminus. The pentraxin family has been implicated in humoral innate immunity. Depending on their structure, pentraxins are divided into short and long pentraxin families. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins, while long pentraxins include pentraxin 3 (PTX3) and neuronal pentraxins [6, 7].

PTX 3 production is stimulated by cytokines and bacterial endotoxins [8]. PTX3 acts as an acute phase reactant protein as its blood levels, which is low in normal conditions (less than 2 ng/ml), increase dramatically to a peak of 200–800 ng/ml within 6–8 hours during endotoxic shock, sepsis and other inflammatory conditions [9].

The aims of our study was to investigate the diagnostic value of PTX 3 in neonatal sepsis.

2 Patients and methods

This prospective case-control study was conducted on 60 neonates diagnosed with culture proven late-onset sepsis and admitted to the neonatal intensive care unit (NICU) at Beni-Suef University Hospital during the period between December 2017 and July 2018. Thirty age- and sex-matched apparently healthy newborns (control group) were recruited from follow-up clinic for healthy neonate. Blood samples were taken at equivalent post-natal age. In control group neonates blood samples were taken simultaneously with scheduled bilirubin level test. The study was approved by the ethics committee at Beni-Suef University Hospital and written informed consent was obtained from infants’ caregivers.

Sepsis was defined as a positive blood culture in newborn with clinical and laboratory findings of infection. Clinical signs include temperature instability, hypotension, prolonged capillary refilling time, tachycardia or bradycardia, apnea, respiratory distress, irritability, lethargy, seizures, poor feeding, vomiting, abdominal distention, jaundice, petechiae and bleeding [10]. Neonates with congenital anomalies, acute renal injury and those exposed to perinatal asphyxia were excluded from the study.

The demographic and perinatal data were collected and included: gestational age, weight, gender, mode of delivery and risk factors of sepsis. Clinical evaluation included signs and symptoms of sepsis. Routine sepsis work up included the following: complete blood count (CBC), C-reactive protein (CRP) as well as blood culture.

2.1 Measurement of PTX3 serum levels

Blood samples for PTX3 measurement were taken from a peripheral vein of septic neonates, as well as from the healthy controls. Then blood samples were immediately centrifuged at approximately 1000×g for 15 minutes to obtain serum and immediately stored at –80°C for further process. Serum levels of centrifuged at approximately 1000 x g were measured by commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (Cusabio Biotech Co., Ltd., China).

2.2 Statistical analysis

Quantitative parametric data were presented as mean and standard deviation (SD), while qualitative (categorical) data were presented as frequencies and percentages. Independent t tests were used to compare continuous variables between cases and control groups. Chi-square tests were used to test association between categorical variables. To measure the strength of the association between PTX3 and other factors, Pearson’s correlation coefficients were used. The significance level was set at it p < 0.05. The receiver operator characteristic (ROC) curve was used to display the relationship between sensitivity and specificity. Data were analyzed using SPSS (Statistical Package for the Social Science, Chicago, IL, USA) version 18.

3 Results

A total of 90 infants were enrolled in our study; 60 with neonatal sepsis and 30 controls. The sepsis and control groups did not differ in their characteristics including sex, mode of delivery, gestational age or weight. Demographic data of the studied population are shown in Table 1. Neonates with sepsis displayed a significantly higher serum level of PTX3 compared to controls (28.98±9.04 μg/L vs. 2.89±0.94 μg/L, p < 0.001).

Table 1
Demographic data of sepsis and control groups

Sepsis group (n = 60) Control group (n = 30) P value

Gender (M/F) 34/26 16/14 0.764

Gestational age (weeks)^* 35.77±2.37 35.6±2.03 0.742

Weight (grams)^* 2860±598 2868±478 0.947

Mode of delivery (CS/NVD) 49/11 23/7 0.576

Post-natal age (days) 7.7±1.72 7.2±1.15 0.194

Hb (gm/dl)^* 10.33±2.43 13±1.78 <0.001

Platelets (10³/μL ^* 99.9±24.6 167.2±32.5 <0.001

WBC (10³/μL)^* 14.98±4.31 11±3.06 <0.001

Pentraxin 3 (μg/L)^* 28.98±9.04 2.89±0.94 <0.001

	Sepsis group (n = 60)	Control group (n = 30)	P value
Gender (M/F)	34/26	16/14	0.764
Gestational age (weeks)^*	35.77±2.37	35.6±2.03	0.742
Weight (grams)^*	2860±598	2868±478	0.947
Mode of delivery (CS/NVD)	49/11	23/7	0.576
Post-natal age (days)	7.7±1.72	7.2±1.15	0.194
Hb (gm/dl)^*	10.33±2.43	13±1.78	<0.001
Platelets (10³/μL ^*	99.9±24.6	167.2±32.5	<0.001
WBC (10³/μL)^*	14.98±4.31	11±3.06	<0.001
Pentraxin 3 (μg/L)^*	28.98±9.04	2.89±0.94	<0.001

^*Values are expressed in mean±standard deviation. CS = cesarean section, NVD = normal vaginal delivery, Hb = hemoglobin, WBC = white blood cells.

The microorganisms identified in blood cultures are shown in Table 2. Klebsiella pneumoniae was the most common organism (26/60) followed by coagulase-negative Staphylococci (12/60). Eighteen of our patients had respiratory distress, however none of them required mechanical ventilation. Five mothers had premature rupture of membranes and 3 mothers had chorioamnionitis while intrauterine growth restriction was found in 6 neonates with sepsis.

Table 2

Distribution of microorganisms isolated from neonates with sepsis

Isolates	Number (%)
Klebsiella pneumoniae	26(43.3)
Coagulase-negative Staphylococci	12(20)
Enterobacter	6(10)
Staphylococcus aureus	5(8.3)
Escherichia coli	4(6.7)
Enterococcus faecalis	2(3.3)
Acinetobacter	2(3.3)
Pseudomonas aeruginosa	2(3.3)
Methicillin-resistant Staphylococcus aureus	1(1.7)

Among neonates with sepsis, 8 neonates (13.3%) died while 52 neonates (86.7%) recovered and discharged. Serum PTX3 level was significantly increased in nonsurvivors when compared to survivors (43.06±3.88 μg/L vs. 26.82±7.52 μg/L, p < 0.001) (Fig. 1).

Fig.1

Pentraxin 3 levels in survivors and nonsurvivors.

There was a significant negative correlation between PTX3 and gestational age (r=–0.471, p < 0.001), platelet count(r=–0.453, p < 0.001) and weight (r = –0.340, p = 0.007), whereas a significant positive correlation was observed between PTX3 and both CRP values (r = 0.773, p < 0.001) and immature to total neutrophil ratio (I/T ratio) (r = 0.726, p < 0.001) (Table 3).

Table 3

Correlation between pentraxin 3 and different studied variables

Parameters	r	p
Gestational age	–0.471	<0.001
Weight	–0.340	0.007
Hemoglobin	0.026	0.844
Platelets	–0.453	<0.001
White blood cells	–0.007	0.958
I/T ratio	0.726	<0.001
CRP	0.773	<0.001

I/T ratio = immature to total neutrophils ratio, CRP = C reactive protein.

ROC curve was generated for PTX3 levels. The characteristics of the ROC analysis (area under the curve: 0.995, 95% CI 0.972–0.999) are given in Fig. 2A. Using a PTX3 cut-off value of 5.6 μg/L for diagnosing sepsis, the sensitivity was 98.3% and the specificity was 96.7% while the positive predicted value (PPV) and the negative predicted value (NPV) were 98% and 96% respectively.

Fig.2

Receiver operating characteristic (ROC) curve between the studied groups (septic group and controls) with regard to PTX3 (A) and CRP (B).

A cut-off value of 8 mg/dl for CRP showed 96.7 % sensitivity and 96.7 % specificity, with positive predicted value (PPV) = 98.3%, negative predicted value (NPV) = 93.5%, and accuracy (area under curve) = 0.989 (Fig. 2B).

4 Discussion

As the clinical manifestations of neonatal sepsis are nonspecific, early diagnosis and treatment still remain a major challenge. Sepsis-specific biomarkers can indicate the presence or absence of sepsis, severity of infection, and can differentiate bacterial from viral and fungal infection [11].

To the best of our knowledge, no previous studies have been undertaken to evaluate the role of PTX3 in diagnosis of neonatal sepsis. In our study, we found a significantly higher level of PTX3 in neonates with sepsis compared to the control group.

PTX3 is a soluble pattern recognition molecule that plays crucial roles in innate immunity. Its principal functions include recognition of microorganisms, activation of complement system and opsonization. Neutrophils serve as PTX3 reservoir, it is stored mainly in specific granules, and partially in azurophilic and gelatinase granules. In response to stimuli, such as microorganisms and inflammatory cytokines (e.g. TNF-α, IL-1β), PTX3 is released into the extracellular space and localized in neutrophil extracellular traps (NETs) [12].

Consistent with the current observations, Hamed et al. [13] found that PTX-3 levels of adult patients with sepsis admitted in ICU were significantly higher than in the control group. Also, a recent study performed by El Gendy et al. [14] has found that PTX3 concentrations were significantly elevated among critically ill septic children compared with controls.

The findings of the present study revealed significantly higher serum levels of PTX3 among non-survivor neonates with sepsis compared to survivor group, indicating that PTX3 may be used as a prognostic marker for neonatal sepsis. In accordance with the present results, previous studies have demonstrated that PTX3 levels remained significantly higher in non-survivors than in survivors [15, 16].

In the current study, the predominant organisms isolated were Gram negative (66.7%), with Klebsiella pneumoniae as the most common pathogen (43.3% of all cases with sepsis). The present findings seem to be consistent with other reports from Egypt [17 –19].

We found a significant negative correlation between PTX3 and both gestational age and weight. Our results share a number of similarities with Akin et al. [20] findings, they found inverse correlation between PTX3 and gestational age but not with the infant’s weight.

One of the major cause of thrombocytopenia in neonates is sepsis, however the exact underlying mechanism is not fully understood. Recent evidence suggests that endothelial damage activates reticule-endothelial removal of platelets. Thrombocytopenia occurs as the rate of peripheral platelet destruction exceeds the rate of bone marrow production [21]. In the current study, platelet count was significantly lower in neonates with sepsis and PTX3 was negatively correlated with platelet count. This was in agreement with Mauri and colleagues who performed their study on 90 adult patients with sepsis and found that PTX3 was significantly related to blood platelets count [15].

The I/T ratio has been widely used for early detection of neonatal sepsis. It has been suggested that I/T ratio is independent of birth weight and gestational age [22]. In the present study, a significant correlation was found between PTX3 and I/T ratio. The observed correlations between PTX3 levels and both I/T ratio and platelet count in neonates with sepsis may indicate that PTX3 levels is associated with the severity of infection.

C-reactive protein (CRP) is the most widely used laboratory tests in the diagnosis of neonatal sepsis. The delayed synthesis during the inflammatory response to infection accounts for its low sensitivity during the early stages of sepsis. Elevated CRP levels have also been observed during numerous non-infectious conditions, thus making it a nonspecific marker for diagnosis of neonatal sepsis [23]. In our study, we found a significant correlation between PTX3 and CRP in neonates with sepsis.

The optimal cut off values of PTX3 for prediction of bacterial infection varied between different studies with variable sensitivities and specificities reported. De Kruif et al. [24] investigated PTX3 levels in febrile adult patients admitted to emergency departments and reported a sensitivity of 76% and specificity of 61% at a cut-off value of 16.1 μg/L. Hamed et al. [13] studied PTX3 levels during days 1, 3, and 8 of ICU treatment and found that the minimal sensitivity and specificity were 92% and 64% respectively at a cut off value of 5 μg/L.

We observed, at a value of 5.6 μg/L, PTX3 has a sensitivity of 98.3%, specificity of 96.7%, PPV of 98% and NPV of 96%.; whereas at a cutoff value of 8 mg/dl, CRP showed 96.7 % sensitivity and 96.7 % specificity. For the diagnosis of neonatal sepsis, the sensitivity of the biomarker is more important compared to its specificity, so PTX3 seem to be superior to CRP as a diagnostic marker for neonatal sepsis.

We aware that our research may have some limitations. First, the small number of the studied patients. Second, single measurement of PTX3 levels was done, so serial measurement of PTX3 might be more useful.

5 Conclusion

PTX3 could be used as a reliable marker of neonatal sepsis with high sensitivity and specificity. PTX3 value higher than 5.6 μg/L should raise the suspicion of sepsis. PTX3 had better sensitivity when compared with CRP. Serum levels of PTX3 were higher in nonsurvivor septic neonates compared to survivors and could be a useful biomarker in predicting outcome of sepsis.

Declaration of interest

The authors have no conflicts of interest relevant to this article.

Source of fund

None

Footnotes

Acknowledgement

The authors would like to thank all their colleagues in the NICU, Beni Suef University Hospital, for their valuable cooperation.

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