Apelin and Procalcitonin in Neonatal Sepsis

Abstract

Introduction: The identification and treatment of sepsis continues to be a major health issue. The incidence of sepsis is particularly high in the neonatal population. Aim of the work: The aim of the present study is to evaluate the role of apelin in neonatal sepsis and its relation with other biomarkers as procalcitonin and blood culture results. Subjects and Methods: This study included 90 neonates divided into two groups: Group I: included 60 neonates with clinically sepsis diagnoised according to (Thaver and Zaidi, 2009) who were subgrouped into group I a (EOS) (early onset ≤ 72h) included 36 neonates and group I b (LOS) (late onset > 72) included 24 neonates. Results: The study was carried out on 90 neonates divided into two groups: Group I : included 60 neonates with clinically sepsis and subgrouped according to onset of sepsis into group I a (early onset ≤ 72h) included 36 neonates and group I b (late onset > 72) included 24 neonates. They were selected from NICU (neonatal intensive care unit) of El-Minia Obstetric and Pediatric University Hospital from February to September 2015. Conclusion: Although blood culture is a gold standerd for diagnosis of neonatal sepsis, but we cannot depend on it only. Apelin and procalcitonin are reliable diagnostic markers of neonatal sepsis which have the same diagnostic accuracy. Apelin and procalcitonin are a good marker for early diagnosis of neonatal sepsis but Apelin is more perfect marker than procalcitonin in diagnosis of early neonatal sepsis. The use of apelin and other markers (procalcitonin, CRP, TLC, platlets and blood culture) collectively yeild the best results for diagnosis of neonatal sepsis.

Full text

MJMR, Vol. 31, No. 2, 2020, pages (59-64). Ali et al.,
59 Apelin and Procalcitonin in Neonatal Sepsis
Research Article
Apelin and Procalcitonin in Neonatal Sepsis
Lamia H. Ali, Ahmed A. Saedii, Mohammed A. Abdel Hakeem
and Enas M. Mostafa Kamal
Department of Clinical Pathology, El-Minia Faculty of Medicine
Abstract
Introduction: The identification and treatment of sepsis continues to be a major health issue. The
incidence of sepsis is particularly high in the neonatal population. Aim of the work: The aim of the
present study is to evaluate the role of apelin in neonatal sepsis and its relation with other biomarkers
as procalcitonin and blood culture results. Subjects and Methods: This study included 90 neonates
divided into two groups: Group I: included 60 neonates with clinically sepsis diagnoised according to
(Thaver and Zaidi, 2009) who were subgrouped into group I a (EOS) (early onset ≤ 72h) included 36
neonates and group I b (LOS) ( late onset > 72) included 24 neonates. Results: The study was carried
out on 90 neonates divided into two groups: Group I : included 60 neonates with clinically sepsis and
subgrouped according to onset of sepsis into group I a (early onset ≤ 72h) included 36 neonates and
group I b (late onset > 72) included 24 neonates. They were selected from NICU (neonatal intensive
care unit) of El-Minia Obstetric and Pediatric University Hospital from February to September 2015.
Conclusion: Although blood culture is a gold standerd for diagnosis of neonatal sepsis, but we
cannot depend on it only. Apelin and procalcitonin are reliable diagnostic markers of neonatal sepsis
which have the same diagnostic accuracy. Apelin and procalcitonin are a good marker for early
diagnosis of neonatal sepsis but Apelin is more perfect marker than procalcitonin in diagnosis of early
neonatal sepsis. The use of apelin and other markers (procalcitonin, CRP, TLC, platlets and blood
culture ) collectively yeild the best results for diagnosis of neonatal sepsis.
Keywords: Apelin, Procalcitonin, Neonatal Sepsis
Introduction
The identification and treatment of sepsis
continues to be a major health issue. The
incidence of sepsis is particularly high in the
neonatal population. The most reliable diagno-
stic test of neonatal sepsis, often referred to as
the gold standard, is a blood culture test for
bacteria. While this test is the most reliable
available, it can take 48 hours to obtain the
results. As a result, treatment must often begin
before the results are known. An additional
complication is the fact that the blood culture
test can be negative for one in five subjects with
sepsis. Thus, it is of critical importance to
identify new biomarkers that will enable fast
and reliable hematological scoring systems for
sepsis in its earliest stages (Kun Wang, et al.,
2013).
Neonatal sepsis is one of the most common
causes of neonatal morbidity and mortality
especially in developing countries. It may be
early onset or late onset according to the
neonatal age at the onset of the infection (Stoll
BJ. et al., 2011). Early onset of neonatal sepsis
is often caused by organism acquired during
delivery while late onset type is due to
organism acquired from nosocomial or
community source (Robinson et al., 2008).
Procalcitonin (PCT) is a 116 amino-acid
peptide precursor of the hormone calcitonin,
which has been proposed as a reliable
diagnostic and prognostic marker of sepsis,
better differentiating inflammatory responses
from bacterial infections. In healthy individuals,
PCT is secreted only in neuroendocrine cells of
the thyroid; however, during an infection, PCT
is released up to a thousand fold increase from
nearly all tissues and cell types in the host in
response to cytokines and bacterial products
(Ravi S. Samraj, et al., 2013).
Apelin is a proinflammatory adipocyte derived
factor that participate in vascular wall inflam-
mation (G.I. Gad, et al., 2014). Apelin
expression is induced by inflammatory media-
tors, such as tumor necrosis factor, interleukin-6
and interferon and plasma apelin levels
correlate with markers of inflammation (Han et

MJMR, Vol. 31, No. 2, 2020, pages (59-64). Ali et al.,
60 Apelin and Procalcitonin in Neonatal Sepsis
al.., 2008) and anti inflammatory end inhibitory
effect on release of inflammatory mediators
which has recently been linked toneonatal
sepsis (ShimingXu, et al., 2012).
Aim of the work
The aim of the present study is to evaluate the
role of apelin in neonatal sepsis and its relation
with other biomarkers as procalcitonin and
blood culture results.
Subjects and Methods
This study included 90 neonates divided into
two groups:
Group I: included 60 neonates with clinically
sepsis diagnoised according to (Thaver and
Zaidi, 2009) who were subgrouped into group I
a (EOS) (early onset ≤ 72h) included 36
neonates and group I b (LOS) ( late onset > 72)
included 24 neonates.
They were selected from NICU (neonatal
intensive care unit) of El-Minia Obstetric and
Pediatric University Hospital from February to
September 2015.
Group II: included 30 appearantly healthy
neonates as a control group.
All neonates were subjected to:
1- Complete history taking and clinical
examination.
2- Laboratory investigations including :
A) Routine investigations :
a- Complete blood count.
b- C-reactive protein.
c- Blood culture for neonates with sepsis
B) Special investigation:
a- Assessment of procalcitonin by enzyme
linked immunoassay (EIA).
b- Assessment of aplien by EIA.
Sampling Protocol:
Under complete sterile conditions, 4ml of
venous blood was drawn from each neonate,
one ml was used in the blood culture firstly then
one ml was used for complete blood count in a
tube containing (K-EDTA) and two ml in plane
tube was left until clotting then centrifuged , the
separated serum was used for CRP and the
remaining serum was stored at -70C till the time
of assessment of procalcitonin and aplien by
EIA .
Laboratory investigations include:
A- Routine investigations:
1- Complete blood count was done using
automated cell counter (Sysmex KX-21-N -
Japan) and blood smear stained by Leishman
stain.
2- C-reactive protein (TECO DIAGNOSTICS
U.S.A.) (Fischel, et al., 1967).
3- Blood culture (conventional method):
One ml of withdrawn venous blood was
inoculated into blood culture bottle, mixed well
then incubated at 370C for 24 hours; subculture
on blood agar and Mac-Conkeys agar plates
every other day was done for ten days.
Further identification for the growing organism
by Gram stained smear and biochemical
reactions were done e.g catalase, coagulase,
triple sugar iron (TSI), citrate and Lysine iron
agar (LIA) then antibiogram were done to
complete sensitivity.
B- Special investigation:
1- Quantitative assay of procalcitonin by
Humareader plus, model 3700, Germany and
kits was supplied by (Ray Bio, U.S.A.):
I- Principle of assay:
The kit was depended upon binding of an
antigen specific for human procalcitonin coated
on a 96-well plate. Standards and samples were
pipetted into the wells and procalcitonin
presented in a sample was bound to the wells by
the immobilized antibody. The wells were
washed and biotinylated anti-human procalci-
tonin antibody was added. After washing away
unbound biotinylated antibody, HRP-conju-
gated streptavidin was pipetted to the wells. The
wells were again washed, a TMB substrate
solution was added into the wells and color was
developed in proportion to the amount of
Procalcitonin bound. The stop solution
changed the color from blue to yellow, and the
intensity of the color was measured at 450 nm
which directly proportional to procalcitonin
concentration. The given standerd concen-
trations were assayed and a curve was drawn
for determination of each sample level.
Results
The study was carried out on 90 neonates
divided into two groups:

MJMR, Vol. 31, No. 2, 2020, pages (59-64). Ali et al.,
61 Apelin and Procalcitonin in Neonatal Sepsis
Group I: included 60 neonates with clinically
sepsis and sub grouped according to onset of
sepsis into group I a (early onset ≤ 72h)
included 36 neonates and group I b( late onset >
72) included 24 neonates.
They were selected from NICU (neonatal
intensive care unit) of El-Minia Obstetric and
Pediatric University Hospital from February to
September 2015.
Group II: included 30 neonates without
apparently healthy without of sepsis.
All different data in studied groups will be
summarized in the following tables:
Table I: Comparison of demographic data in both groups
Group I
Neonatal sepsis
(n=60)
Group II
Control
(n=30)
P value
Age (days)
Range
Mean ± SD
(1-27)
7.96±5.82
(1-24)
8.33±4.52
0.854
Sex
Male
Female
33(55%)
27(45%)
22(73.3%)
8(26.7%)
0.197
Gestational age
Preterm
Fullterm
36(60%)
24(40%)
14(46.7%)
16(53.3%)
0.350
Mode of delivery
SVD
CS
23(38.3%)
37(61.7%)
14(46.7%)
16(53.3%)
0.556
- *: significant difference at p value < 0.05
As shown in table (I) the age in group I was
ranged from 1day to 27 days with a mean ± SD
of 7.96±5.82. While the age ranged from 1 day
to 24 days with a mean ± SD 8.33±4.52 in
group II . There was no significant difference
between both groups regarding the age (P value
= 0.854).
Sex: group I included 33 males (55%) and 27
females (45%) while group II included 22
males (73.3%) and 8 females (26.7%) with no
significant difference between both groups
regarding to the gender(P value = 0.197).
Gestational age: group I included 36 preterm
neonates (60%) and 24 full term neonates
(40%) while group II included 14 preterm
neonates (46.7%) and 16 full term neonates
(53.3%) with no significant difference between
both group regarding to the gestational age ( P
value = 0.350).
Mode of delivery: group I included 23
neonates were delivered by spontanous vaginal
delivery (38.3%) and 37 neonates were deliv-
ered by cesserian section (61.7%) while group
II included 14 neonates were delivered by
spontanous vaginal delivery (46.7%) and 16
neonates were delivered by cesserian section
(53.3%) with no significant difference between
both group regarding to mode of delivery ( P
value = 0.556).

MJMR, Vol. 31, No. 2, 2020, pages (59-64). Ali et al.,
62 Apelin and Procalcitonin in Neonatal Sepsis
Table II: Comparison between both groups regarding TLC, Hb, Platelets count and CRP
Group I
Neonatal sepsis (n=60)
Group II
Control (n=30)
P value
TLC (c/mm3)
Range
Mean ± SD
(1600-21900)
11540±4932.84
(4100-6500)
4926.66±698.43
< 0.001*
Hb(g/dl)
Range
Mean ± SD
(8.8-19.9)
14.22±2.58
(14-18.6)
16.14±1.38
< 0.001*
Platelets (/mm3)
Range
Mean ± SD
(18.000-350.000)
196.380±94.230
(198.000-365.000)
267.330±53.170
< 0.001*
CRP
-Ve
17(28.3%)
30(100%)
< 0.001*
- *: significant difference at p value < 0.05
Table (II) showed the results of total leucocytic
count (TLC), haemoglobin, platelets and C-
reactive protein . TLC count in group I ranged
from 1600 to 21900/mm3 and the mean ±SD
was 11540± 4932.84 while in group II it
ranged from 4100 to 6500/mm3 and the mean
±SD was 4926.66± 698.43. There was a high
statistically significant increase in TLC in group
I comparing with group II (P value <0.001).
Haemoglobin in group I ranged from 8.8 to 19.9
gm/dl and the mean ±SD was 14.22 ± 2.58
while in group II it ranged from 14 to 18.6
gm/dl and the mean ±SD was 16.14 ±1.38.
There was a high statistically significant
decrease in Hb in group I when compared with
group II (P value <0.001).
Platelets in group I ranged from 18.000 to
350.000/mm3 and the mean ±SD was 196.380±
94.230 while in group II ranged from 198.000
to 365.000/mm3 and the mean ±SD was
267.330± 53.170. There was high statistically
significant decrease between two groups (P
value =0.001).
CRP: Group I included 17 neonates with
negative CRP (28.3%) while group II included
30 neonates with negative CRP (100%). There
was a high statistically significant difference
between two groups (P value <0.001).
Table III: Frequency of microorganisms in blood cultures of neonatal sepsis group
Blood culture
Group I
Neonatal sepsis (n=60)
Blood culture
-Ve (no growth)
+Ve
28(46.7%)
32(53.3%)
Staphylococcus aureous
7(11.7%)
Staphylococcus epidermidis
5(8.3%)
E. Coli
5(8.3%)
Enterobacter
4(6.7%)
E.Coli Kelebsiella
3(5%)
Streptococcus pyogens
2(3.3%)
NH streptococci
2(3.3%)
Staphylococcus saprophyticus
1(1.7%)
Pseudomonous
1(1.7%)
Proteous
1(1.7%)
Candida
1(1.7%)

MJMR, Vol. 31, No. 2, 2020, pages (59-64). Ali et al.,
63 Apelin and Procalcitonin in Neonatal Sepsis
Discussion
Neonatal sepsis is one of the most common
causes of neonatal morbidity and mortality
especially in developing countries. It may be
early onset or late onset according to the
neonatal age at the onset of the infection (Stoll
BJ. et al., 2011). Early onset of neonatal sepsis
is often caused by organism acquired during
delivery while late onset type is due to
organism acquired from nosocomial or
community source (Robinson et al., 2008).
Early diagnosis of neonatal sepsis and interv-
ention are essential to avoid serious compli-
cation (fatal organ failure and death) (Tang et
al., 2007). The most reliable diagnostic test of
neonatal sepsis, often referred to as the gold
standard, is a blood culture test for bacteria.
While this test is the most reliable available, it
can take 48 hours to obtain the results. As a
result, treatment must often begin before the
results are known. An additional complication
is the fact that the blood culture test can be
negative for one in five subjects with sepsis.
Thus, it is of critical importance to identify new
biomarkers that will enable fast for sepsis in its
earliest stages (Kun Wang et al., 2013).
In this work, a total number of this study was
60 septic neonates which were admitted to
NICU at Minia University Hospital from the
period of February to September 2015
according to clinical mainfestation as in a study
done by Wynn J. et al., 2010. Out of this 60
septic neonates, 36 cases were diagnosis as
EOS (60%) and 24 cases as LOS (40%). The
results were in agreement with Gad GI. Et al.,
2014 and Katherine Soreng, and H. Roma Levy,
2011.
The study included 23 cases (38.7%) were
delivered by normal vaginal delivery and 37
cases (61.7%) were born by CS showing higher
sepsis rate among neonates born by CS and this
was in concordance with studies done by Aaron
B. Caughey MD. et.al, 2014 reported that CS
was more safe and decreased risk of sepsis and
mortality rate and this may be explained by
roles of infection control done perfectly in
U.S.A.
In the present study, total leucocytic count in
septic neonates was increase when compared
with control group (P value <0.001) that means
it a good indicator of sepsis although it was a
wide range in count so (range from 1600 to
21900/mm3), it is of little clinical use in
diagnosis of neonatal infection because of wide
variation in values. These results were in
agreement with Khair et.al., 2010 who reported
that TLC was not increased in all cases and its
sensitivity was 50%. It remains nonspecific and
have a low positive predictive value. In our
study there were 38 neonates with normal TLC
counts in group I which means we cannot
depend on TLC count only. This was in
agreement with a study done by Khashu M. et
al., 2006reported that normal TLC counts may
be initially observed in as many as 50% of
cases of culture-proven sepsis.
Haemoglobin level in septic neonates was
decreased when compared with control group
(P value <0.001) these results were in
agreement with that obtained by Ryon M. et
al., 2017 who reported that the effects of sepsis
on the erythrocyte, including changes in RBC
volume, metabolism and hemoglobin’s affinity
for oxygen, morphology, RBC deformability
(an early indicator of sepsis), antioxidant status,
intracellular Ca2+ homeostasis, membrane
proteins, membrane phospholipid redistribution,
clearance and RBC O2-dependent adenosine
triphosphate efflux (an RBC hypoxia signaling
mechanism involved in micro vascular auto
regulation) and also consider the causes of these
effects by host mediated oxidant stress and
bacterial virulence factors.
As for platelets counts in my study, there were
markedly lower in neonates with sepsis than in
control group with ( P value < 0.001) which
means that thrombocytopenia is one of the
laboratory markers for sepsis and this was in
agreement with a study done by Khair et al.,
2010 who found that neonates with sepsis
develop thrombocytopenia, possiblly because of
the damaging effects of endotoxin on platletes.
Conclusion
1- Although blood culture is a gold standerd
for diagnosis of neonatal sepsis, but we
cannot depend on it only.
2- Apelin and procalcitonin are reliable
diagnostic markers of neonatal sepsis
which have the same diagnostic accuracy.
3- Apelin and procalcitonin are a good
marker for early diagnosis of neonatal
sepsis but Apelin is more perfect marker

MJMR, Vol. 31, No. 2, 2020, pages (59-64). Ali et al.,
64 Apelin and Procalcitonin in Neonatal Sepsis
than procalcitonin in diagnosis of early
neonatal sepsis.
4- The use of apelin and other markers
(procalcitonin, CRP, TLC, platlets and
blood culture) collectively yeild the best
results for diagnosis of neonatal sepsis.
References
1. Aaron B.Caughey, Alison G.Cahill,
Jeanne-MarieGuise and J.Rouse (2014).
Safe prevention of the primary cesarean
delivery. Vol 210. 179-193.
2. Adam SS, Key NS and Greenberg CS
(2009). D-dimer antigen: current concepts
and future prospects. Blood; 113:2878–
2887.
3. Adams-Chapman, Ira Stoll and Barbara J
(2006). Neonatal infection and long-term
neuro developmental outcome in the
preterm infant, Vol 19: 3, 290–297.
4. Adib M, Bakhshiani Z, Navaei F,
SahebFosoul F, Fouladi S and
Kazemzadeh H (2012). Procalcitonin: a
reliable marker for the diagnosis of
neonatal sepsis. Iran J Basic Med Sci.; 15:
777–782.
5. Agnieszka M. Tycinska, Anna Lisowska,
Wlodzimierz J. Musial and Bozena
Sobkowicz (2012). Apelin in acute
myocardial infarction and heart failure
induced by ischemia; vol 7 (408-412).
6. Alan G. Japp and David E (2008). Newby
The apelin–APJ system in heart failure
Pathophysiologic relevance and therapeutic
potential, 1882 – 1892.
7. Alcaide P, Auerbach S and Luscinskas FW
(2009). Neutrophil recruitment under shear
flow: It’s all about endothelial cell rings
and gaps. Microcirculation;16:43–57.
8. Ali AM, Elkhatib WF and Abdelaziz SS
(2014). Procalcitonin versus C-Reactive
Protein in Neonatal Sepsis. Journal of
Immunology and Infectious Diseases. vol
1, 2394-6512.
9. Alison Bedford Russell (2011). Paediatrics
and Child Health (6):265–269.
10. Altunhan H, Annagur A, Ors R and
Mehmetoglu I (2011). Procalcitonin meas-
urement at 24 hours of age may be helpful
in the promptdiagnosis of early-onset
neonatal sepsis. International Journal of
Infectious Disease;15:e854-e858.
11. Atluri P, Morine KJ, Liao GP, Panlilio
CM, Berry MF and Hsu VM (2007).
Ischemic heart failure enhances endo-
genous myocardial apelin and APJ receptor
expression. Cellular & molecular biology
letters; 12:127–38.
12. Cagno, C.K., Pettit, J.M., and Weiss, B.D
(2012). Prevention of perinatal group B
streptococcal disease: updated CDC guide-
line. Am Fam Physician; 86: 59–65
13. Committee on Infectious Diseases,
Committee on Fetus and Newborn, Baker,
C.J., Byington, C.L., and Polin, R.A
(2011). Policy statement—Recommen-
dations for the prevention of perinatal
group B streptococcal (GBS) disease.
Pediatrics.128: 611–616.
14. Fukushima H, Kobayashi N, Takeshima H,
Koguchi W and Ishimitsu T (2010). Effects
of olmesartan on Apelin/APJ and
Akt/eNOS pathway in Dahl rats with end-
stage heart failure. J CardiovascPharmacol.
Jan;55(1):83–8.
15. Han S, Wang G, Qi X, Englander EW, and
Greeley GH Jr (2008) Involvement of a
Stat3 binding site in inflammation-induced
enteric apelin expression. Am J Physiol
Gastrointest Liver Physiol 295:G1068–78.
16. Jiang, Z. and Ye, G.Y.(2013). 1:4 matched
case-control study on influential factor of
early onset neonatal sepsis. Eur Rev Med
Pharmacol Sci; 17: 2460–2466.
17. Kun Wang, Vineet Bhandari, Sofya
Chepustanova, Greg Huber, Stephen
O′Hara, Corey S. O′Hern, Mark D.
Shattuck, and Michael Kirby (2013).
Which Biomarkers Reveal Neonatal
Sepsis? PLoS One 8(12): e82700.
18. M. J. Hill, S. Kumar and P.G. McTernan
(2009). Adipokines and the clinical labo-
ratory; what to measure, when and how?
J.Clin. Pathol., 62: 206- 211.
19. Metsvaht T, Pisarev H, Ilmoja3 M-L, Parm
U, Maipuu L, Merila M, Müürsepp P and
Lutsar I (2009). Clinical parameters
predicting failure of empirical antibacterial
therapy in early onset neonatal sepsis,
identified by classification and regression
tree analysis BMC Pediatrics, 9:72.
doi:10.1186/1471-2431-9-72.
20. Nakamura, A., Wada, H., Ikejiri, M.,
Hatada, T., Sakurai, H., Matsushima Y, et
al., (2009). Efficacy of procalcitonin in the
early diagnosis of bacterial infections in a
critical care unit. Shock.; 31: 586–591.