# Serum Amyloid A, Procalcitonin, Tumor Necrosis Factor-α, and Interleukin-1β Levels in Neonatal Late-Onset Sepsis

Authors: Birsen Ucar; Bilal Yildiz; M. Arif Aksit; Coskun Yarar; Omer Colak; Yildiz Akbay; Ertugrul Colak
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Background. Sepsis is an important cause of mortality in newborns. However, a single reliable marker is not available for the diagnosis of neonatal late-onset sepsis (NLS). The aim of this study is to evaluate the value of serum amyloid A (SAA) and procalcitonin (PCT) in the diagnosis and follow-up of NLS.
Methods. 36 septic and healthy newborns were included in the study. However,
SAA, PCT, TNF-α, IL-1β, and CRP were serially measured on days 0, 4, and 8 in the patients and once in the controls.
Töllner's sepsis score (TSS) was calculated for each patient. Results. CRP, PCT, and
TNF-α levels in septic neonates at each study day were significantly higher than in the controls (P=.001).
SAA and IL-1β
levels did not differ from healthy neonates. The sensitivity and specificity were
86.8% and 97.2% for PCT, 83.3% and 80.6% for
TNF-α, 75% and 44.4%
for SAA on day 0. Conclusion. Present study suggests that CRP seems to be the most helpful indicator and PCT and
TNF-α may be useful markers for the early diagnosis of NLS. However,  SAA, IL-1β, and TSS are not reliable markers for the diagnosis and follow-up of NLS.


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## Body

## 1. Introduction

Sepsis and septic shock in newborn infants have a high risk of
morbidity and mortality. Despite advances in medicine, diagnosis of neonatal
sepsis remains as a major challenge. Early clinical signs are
nonspecific and the laboratory criteria arealso not fully reliable.
Warning signs and symptomsare often subtle and can easily be
confused with noninfective causes such as apnea, hypothermia, and
acute exacerbation of chroniclung disease. So that hematological
and biochemical markers such as immature/total neutrophil ratio,
platelet count, C-reactive protein (CRP), various cytokines,
procalcitonin (PCT), and tumornecrosis factor-α (TNF-α)
have been proposed as being useful indicators for early
identification of septic infants [1–4].
Recently, SAA that refers to a group of polymorphic apolipoproteins 12–14 kDa, mainly
produced by the liver, has been proposed as a new discriminative marker of
bacterial infection [1, 3–6]. But the results of these rare studies are contradictory. In this study, we investigated
the value of SAA and procalcitonin levels in the diagnosis and followup of
neonatal late-onset sepsis (NLS).


## 2. Material and Methods

Thirty six newborn infants who were diagnosed as
having clinical suspected NLS in the neonatal intensive care unit at the medical
faculty of Eskisehir Osmangazi University
between June 2003 and June 2004 were included in this prospective study.
Thirty six healthy newborns who had normal clinical and laboratory findings
were included as a control group. They were selected from the neonatal unit or
well-baby outpatient clinic. Diagnosis of sepsis was done according to the 2001
International Sepsis Definitions Conference criteria. According to this
conference, diagnostic
criteria for sepsis in the pediatric population are signs and symptoms of
inflammation including arterial hypotension-decreased capillary refill or
mottling cardiac index >5.5, significant edema or positive fluid balance,
ileus plus infection (documented or suspected) with hyper or hypothermia,
tachycardia, and at least one of the following indications of altered organ
function: altered mental status, acute oliguria, hypoxemia, increased serum
lactate level, or bounding pulses [7]. Laboratory criteria were
leukocytosis, leukopenia, immature leukocyte count >10%, and high CRP levels
[7].Töllner's sepsis score (TSS) was calculated for each
patient [8]. This scoring system consists of clinical (skin color, body
temperature, muscle tonus, breath rate, abdominal distension, imperfect
microcirculation, and risk factors) and laboratory (leukocyte and thrombocyte
counts, CRP, immature/total neutrophil ratio) parameters. A point was given
for each parameter (0, 1, 2, or 3) in respect of their worsening. For example,
0 for normal muscle tonus, 1 for hypotonia, 2 for flask tonus, 0 for normal
leukocyte count, 1 for leukocytosis, and 3 for leukopenia. According to this
scoring system, patients who have >10 points were determined as having
sepsis. Chest radiographs were routinely performed. Hematological and biochemical
markers including a complete blood count, differential white cell count, and
levels of CRP, TNF-α, IL-1β, PCT, and SAA were serially measured. The initial (2 mL) blood samples were
obtained from patients on day 0 (at the time of sepsis diagnosis). Two further
samples were obtained from each patient on days 4 and 8 for follow-up in the
patient group. However, only one blood sample (2 mL) was obtained from each healthy
control subject.Serum CRP levels were measured by radio-immuno-assay
method (cut-off level: 0.8 mg/dL). TNF-α (cut-off level: 1 pg/mL), IL-1β, and SAA (cut-off level: 6610 ng/mL) levels were measured by ELISA (BioSource
International Immunoassay, BioSource International, Inc., Calif, USA).
PCT (cut-off level: 0.8 ng/mL) levels were analyzed by immunoluminometric
method (Brahams PCT LUMItest, BRAHAMS-Hennigsdorf, Berlin, Germany).
Blood specimens for TNF-α, IL-1β, PCT, and SAA were
immediately immersed in ice and transported to the laboratory for processing.
Serum was separated by centrifugation at 4°C and stored in 200 μL aliquots at −72°C until analysis.The receiver operator characteristic (ROC) method was
used to determine the best cut-off point of CRP, TNF-α, PCT, and SAA.The study was approved by the Research Ethics Committee of the Faculty
of Medicine, Eskisehir Osmangazi University.
Informed consents were obtained from the parents or guardians for all study
patients and control subjects.Statistical analyses were performed using SPSS
10.0.χ2, post hoc and Mann Whitney-U tests were used to compare categorical predictors.
Also, multivariate analyses were performedfor logistic regression
analysis. The sensitivity
and specificity were calculated for days 0, 4, and 8 to construct the receiver
operating characteristic curves for each biochemical marker.


## 3. Results

The demographic characteristics of the study and control groups are
summarized in Table1. There were no significant differences between the
two groups for gestational age, sex, birth weight, or Apgar scores at 1 and 5
minutes. Clinical and laboratory findings of the study group are given in Table
2. Hypotonia, changes in body temperature, cutis marmoratus,
dyspnoea, and hepatomegaly were dominant findings in patients with sepsis. Platelet counts (Mean ± SEM) and I/T ratios (Mean ± SEM) were higher
in patients than in controls (46777.8±10720.6/mm3 versus 215000±47600/mm3; P<.001
and 0.2±0.1 versus 0.002±0.004; P<.001; resp., but leukocyte counts (Mean ± SEM) were similar in patients
and controls 19294.4±8598/mm3 versus 46777.8±10720.6/mm3; P>.05).Table 1
Demographic findings in patients and control subjects*.
SexBirth weightDeliveryAge5′ApgarGirlsBoys(mean ± SD)SVDC/SGestationalPost natalscoren (%)n (%)(g)n (%)n (%)(mean ± SD) (week)(mean ± SD) (day)meanPatients group13 (36.1)23 (63.8)2281.9±127.118 (50)18 (50)34.5±0.56.9±0.69.2Control  group12 (33.3)24 (66.6)2202.1±122.620 (56)16 (44)33.4±0.48 ± 0.88.9SVD: Spontan vaginal delivery; C/S: caesarean section.*P>.05.Table 2
Clinical and laboratory findings in patients.
n%A. Clinical parametersDepressed newborn reflexes3083.3Hypotonia3186.1Change of body temperature2672.2– High (>38°C)1952.8– Low (<36°C)719.4Dyspnoea2672.2Cutis marmoratus2363.9Hepatomegaly2158.3Central cyanosis2055.6Apnea1850Jaundice1541.7Convulsion822.2Lethargy513.9Sclerema513.9B. Laboratory parametersDisseminated intravascular coagulation (DIC)513.9Toxic granulation3391.7High I/T ratio1952.8Blood culture2672.2– Coagulase-negative staphylococci25.6– Staphylococcus aureus411.1– Klebsiella pneumoniae1336.1– Acinetobacter
SPP25.6– Pseudomonas aeruginosa12.8– Enterobacter cloaca25.6– Candida tropicalis12.8– Streptococcus viridans12.8Platelet count (mean ± SEM, per mm3)46777.8±10720.6Leukocyte count (mean ± SEM, per mm3)19294.4±8598During the study
period, 28 (77.8%) patients
were treated with mechanical ventilation. Eight out of these 28 (28.6%) patients had
respiratory dystress syndrome and were treated with surfactant. Thirty of 36 (83.3%)
patients were given oxygen therapy. None of the patients had been treated with pre-/postnatal
antibiotics or steroids and none of them had been operated prior to the
inclusion to the study. Also none of the patients had a central line.The ratio of blood culture-proven septic patients among the study
group was 72.2%. Klebsiella pneumonia was the most prevalent microorganism in the
septic patients.Individual serum levels of PCT, SAA, CRP, TNF-α, and IL-1β in
the whole patient group and control group on days 0, 4, and 8 are shown in
Figure 1. However, Table 3 demonstrates serum levels of acute phase reactants
in the patients with proven sepsis and controls. The serum levels of CRP, PCT,
and TNF-α in the whole patient group on day 0 were found to be significantly
greater than the controls (Figure 1, P=.001). Also, serum levels of CRP, PCT, and TNF-α
in the patients with proven sepsis on day 0 were found to be significantly
higher than in controls. These high levels were continued
on days 4 and 8. On the other hand, serum levels of IL-1β were not higher in
the patient group on days 0, 4, and 8 than the control group. The mean levels
of SAA of the study group were found to be greater than that of the control
group but the difference was not statistically significant (P>.05).Table 3
Serum levels of acute phase reactants in newborns with culture-proven sepsis and
controls (mean ± SD).
PatientsControlsDay 0Day 4Day 8Procalcitonin (ng/mL)89.7±30.3***50.7±16.8***62.7±18.7****1.5±0.1TNF-α (pg/mL)161.9±78.4*86.7±28.6***144.8±58.2**9.3±4.4IL-1β (pg/mL)<1<1<1<1SAA (ng/mL)5529.6±375.55671.5 ± 3675643.5±436.25461.1±407.5CRP (mg/dL)10.4±1.1***8.4 ± 1***7.3±1.1***0.8±0.1*P<.05; **P<.01; ***P<.001; ****P<.0001.Serum levels of CRP, PCT, TNF-α, and SAA on days 0, 4, and 8.
(a)(b)(c)(d)While PCT levels on day 0 were
significantly higher than on day 4 (P<.05), there were no significant
differences between PCT levels on day 4 and day 8 or day 0 and day 8 (P>.05).
TNF-α and SAA levels did not change statistically during the study period (P>.05).
CRP levels on day 0 were significantly higher than on day 4 and day 8 (P=.05
and P=.01, resp.). However, CRP levels on day 4 and day 8 were
similar (P>.05).TSS did not correlate with the serum levels of PCT,
SAA, CRP, TNF-α, IL-1β, or hematological values including I/T ratio, leukocyte,
and thrombocyte counts (P>.05). Also, the serum levels of PCT, SAA,
CRP, TNF-α, or IL-1β did not correlate with platelet counts (P>.05).The serum levels
of CRP negatively correlated with the levels of SAA and positively correlated
with TNF-α on day 0 (r=−0.532, P<.001; r=0.393, P<.05, resp.)
and with the levels of SAA on day 4 (r=−0.481, P<.01). In addition,
the levels of SAA on day 0 positively correlated with the ratio of I/T (r=0.40, P<.05).Multiple
regression analysis showed that the most important factor which influenced the
levels of CRP on days 0, 4, and 8 was TNF-α level (r=0.896, P=.001;
r=0.621, P<.05; r=0.634, P<.01, resp.). The sensitivity
and specificity of PCT, SAA, CRP, and TNF-α for determining sepsis are summarized in Table
4. The sensitivity and specificity of the acute phase reactants on day
0 were CRP (97.2%; 95% CI: 85.4–99.5 and 100%;
95% CI: 100.0-100.0), PCT (86.1%; 95% CI: 70.5–95.3 and 97.2%; 95% CI: 85.4–99.5), TNF-α (83.3%; 95% CI: 67.2–93.6 and 80.6%;
95% CI: 64.0–91.8), SAA (75%;
95% CI: 57.8–87.9 and 44.4%;
95% CI: 27.9–61.9, resp.). Based on these analyses, the most sensitive
parameter was CRP on day 0. Other sensitive parameters were PCT, TNF-α, and
SAA, respectively, on day 0.Table 4
The sensitivity and specificity of acute phase reactants.
Sensitivity (%)Specificity (%)Day 0Day 4Day 8Day 0Day 4Day 8Procalcitonin86.183.369.497.286.197.2TNF-α83.380.686.180.680.680.6CRP97.2100.0100.0100.0100.0100.0SAA75.086.119.444.433.3100.0The ROC curves of PCT, SAA, CRP, and TNF-α are seen in Figure 2.ROC curves and cut-off levels of CRP, PCT, TNF-α, and SAA.
(a)(b)(c)Three patients (8.3%) died due to systemic inflammatory response syndrome. One of them had hydrops fetalis and two
others had respiratory dystress syndrome and also all these three patients had disseminated
intravascular coagulation (DIC). Interestingly, we did not find any significant
increase in the levels of TNF-α and SAA in this patients in comparison to those
who survived, but high levels of PCT were found in these patients on days 0, 4,
and 8 than the control group (90 ± 7.8/1.5±0.1; 42.2±8.3/1.5±0.1;
56.3±7.2/1.5±0.1, resp., P<.001).


## 4. Discussion

Culture-proven
bacterial infections in newborn infants are associatedwith
substantial morbidity and mortality. However, a single reliable biochemical
marker is not available for the diagnosisof neonatal sepsis. SAA
has been proposed for early diagnosis of NLS [4, 5, 9, 10]. Arnon et al.
reported that SAA had an overall better diagnostic accuracy for predicting
early onset sepsis than CRP (sensitivity (96% versus 30%), specificity (95% versus
98%) [4]. Also, they found that SAA was a useful inflammatory marker during
late-onset sepsis in preterm infants [5]. In contrast to these studies in our
study, SAA levels were greater in patients with late-onset neonatal sepsis than
in the control group on days 0, 4, and 8; but this difference was not
statistically significant (P>.05). These results may explain
defective IL-1β production in newborn. In fact, it has been reported that SAA gene
transcription is induced mainly by glucocorticoids, IL-1, IL-6, and TNF-α [11]. However,
Gabay et al. [12] reported that IL-1 and IL-6 but not TNF-α could induce SAA
protein synthesis in human hepatocytes and that IL-6 was the most effective
inducer but synergy with either IL-1 or TNF-α was not seen [12]. Our
experience showed that IL-1 does not increase in septic newborns and TNF-α seems not to
influence the production of SAA in septic newborns. Therefore, the
production of SAA may not be adequately stimulated in newborn. Like IL-1, we do
not propose the use of SAA as an acute phase reactant in NLS.Although, in recent years, severalnew markers ofinfectionhave been
investigated, some studies suggested that CRP remains to be the best diagnostic
test for neonatal sepsis. Especially serial measurements of CRP are highly
specific and sensitive in newborn sepsis [12–16]. Also, our
results showed that CRP is the best sensitive and specific acute phase reactant
for diagnosis of NLS.Our data
indicated that the plasma concentrations of IL-1β
in infected infants were remarkably low and IL-1β was a less satisfactory marker. It seems that the monocytes of newborn
infants may be unable tosecrete adequate IL-1β and prostaglandin E2 (fetal or
maternal) and IL-6 may suppress IL-1β and TNF-α production in infections [17–20]. Therefore,
we do not propose the use of IL-1β as an acute phase reactant in NLS.TNF-α is one of the primary agents which sets in
motion the exaggerated cellular, metabolic, and vascular responses of sepsis
[21–23]. Its
usefulness asa diagnostic marker has not been found to be as good
as either SAA or IL-1 in other studies and the sensitivity and
specificity have been controversial in neonatal sepsis [13, 17, 24]. In one study, the sensitivity and specificity
were found to be 87.9% and 43%, respectively, with high positive and negative
predictive values [17]. In our study, it was found that the sensitivity and specificity of TNF-α
were 83.3% and 80.6%, respectively, on day 0. We found that the
levels of TNF-α positively correlated with the serum levels of CRP on day 0.
So that TNF-α is also a useful marker with
high sensitivity and specificity for the detection of NLS.A few new of markers including PCT seem to be promising as a
diagnostic test for sepsis [3, 6, 25–29]. Some studies
found that the sensitivity of PCT is low (70%–80%) to rule out sepsis at birth
[25, 28]. Indeed, Enguix et al. [6] reported that PCT, CRP, and SAA are
similar diagnostic markers of sepsis in critically ill neonates. In contrast to
these reports, in our study, the order of the markers according to sensitivity
and specificity for optimum prediction of neonatal sepsis is CRP >
PCT > TNF-α> SAA at the time of diagnosis. Therefore, we found that PCT
was the second useful diagnostic marker in sepsis after CRP.Our study confirmed previous findings that neonates
with bacterial sepsis have reduced thrombocyte
count, high I/T ratio and presence of toxic granulation in granulocytes [28, 29], but leukocyte counts were not found to be increased in our study. Also we found that there was not any
correlation between TSS and acute phase reactants or hematological
parameters. Caksen et al. [30] reported that
there was not a significant difference for leukocyte counts, cytokine levels,
and TSS between the blood culture-positive and -negative groups in septic
newborns. We consider that Töllner sepsis score is itself not helpful in
early diagnosis of neonatal bacterial infections.In conclusion, our data indicate that CRP is the best
reliable marker of inflammation for the diagnosis of NLS. PCT may also be used
as a sensitive and specific diagnostic marker in NLS. The SAA did not increase
in early phase in our patients, therefore it cannot be used alone for the
diagnosis and followup of NLS. Also, TSS is not beneficial in late-neonatal
sepsis.



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