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EVALUATION OF 16S rDNA AMPLIFICATION BY PCR AND SOME
IMMUNOLOGICAL MEDIATORS ASSESSMENT COMPARED WITH
BLOOD CULTURE IN DIAGNOSIS OF NEONATAL SEPSIS
By
Mahmoud Shokry*, Mohamed I. Bassyouni*, Sahar Aou-El-Eoon*,
Mohi Moaz* and Samir Tamer**
Departmenets of *Microbiology & Immunology and **Pediatrics,
El-Minia Faculty of Medicine
ABSTRACT:
Background: eonatal sepsis is one of the most important problems in NICUs all over
the world. It is also an important cause of morbidity and mortality in neonates.
Objective: The study aims to identify the common pathogens responsible for
neonatal sepsis at NICU of Minia University Hospital and to evaluate the diagnostic
role of assessment of the immunological mediators; IFNγ, soluble CD14, interleukin6 and CRP in the diagnosis of neonatal sepsis. The study aims also to evaluate the role
of PCR in the diagnosis of sepsis.
Methods: The study was done on 90 infants at NICU of Minia University Hospital.
The study included two groups; septic group composed of sixty infants who were
diagnosed clinically to have neonatal sepsis and co thirty infants excluded to have
sepsis at the clinical level. Blood cultures were done for cases and controls the
isolated bacteria were identified by traditional laboratory methods. The serum levels
of soluble CD14, gamma interferon and interleukin-6 was measured by enzyme linked
immunosorbent assays (ELISA). Also amplification of 16s rDNA gene was performed
by PCR technique.
Results: Of the 60 episodes of clinically diagnosed neonatal sepsis, 54% of the blood
cultures were positive by while 46% were negative. The most common causative
organisms were Coagolase negative staphylococci (31.25%) Staphylococous aureus
(28.1%) Klebsiella (6.25%) and GBS (3.1%) Pseudomonas (6.25%) Escherichia coli
(21.9%). Among laboratory tests, IL-6 had the best sensitivity (91.7%) and negative
predictive value (82.75%), positive predictive value (90.16), the specificity of it was
80%. combined measeurment of crp and/or IL-6 gave the best sensitivity (96.7)
Infants with sepsis were more likely to have apnea/bradycardia (p = 0.002) lethargy
p = 0.0001. From the 60 cases, 56 were PCR positive for 16s rRNA gene with
sensitivity 93.3% specificity 90%, positive predictive value (PPD) 95% and negative
predictive value NPD 85%. One of the four PCR negative was culture positive
KEY WOPRDS:
16S rDNA
Blood Culture
Immunological Mediators
Neonatal Sepsis
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investigated intensively in the past
with
respect
to
developmental
deficiencies of the host defense system,
which include a delayed maturation of
the specific humoral and cellular
immune response of neonatal B and T
cells. Moreover, compromised functions of the innate immune system with
a defective activation of the complement cascade have been described
(Lewis & Wilson, 1995). The
decreased capability of neonatal cells
to secrete cytokines like tumor necrosis
factor
alpha
(TNF-α),
gamma
interferon (IFN-γ), interleukin-1ß (IL1ß), IL-6, IL-8, and IL-12, was
considered as a predisposing factor
increasing incidence of neonatal sepsis
(Berner et al., 2002). However, for
neonatal sepsis, little is known about a
group of molecules playing a central
role in the innate immune system.
Among them are the myeloid antigen
CD14, which is involved in the
recognition of a wide variety of
bacterial products (Medvedev et al.,
1998), and lipopolysaccharide-binding
protein (LBP), which is the principal
plasma protein responsible for
transporting endotoxin to immune
effector cells bearing CD14 on their
cell surfaces (Opal, 1999). Molecular
techniques such as PCR have been
used successfully to identify a wide
range of organisms, including bacteria,
yeasts, viruses, and protozoa (McCabe
et al., 1995). Recently, bacterial DNA
consensus sequences, e.g., the 16S
rRNA gene, have been identified to
define an organism as a bacterium.
With such sequence information
available, numerous DNA primers and
probes have been described for use in
PCR-based assays to diagnose bacterial
sepsis (Laforgia et al., 1997). Unlike
culture, most molecular assays are
designed specifically for one organism.
This provides high sensitivity and
specificity but detects only what you
are looking for; multiple assays may be
INTRODUCTION:
Neonatal sepsis is one of the
most common reasons for admission to
neonatal units in developing countries.
It is also a major cause of mortality in
both developed and developing
countries (Dawodu et al., 1997).The
spectrum of organisms that cause
neonatal sepsis changes over time and
varies from region to region. It can
even vary from hospital to hospital in
the same city (Rahman et al., 2002).
The clinical signs are nonspecific and
indistinguishable from those caused by
a variety of neonatal noninfective
disorders, such as aspiration syndrome,
maladaptation, and respiratory distress
syndrome. It is therefore recommended
for all neonates who develop these
signs to start empirical antimicrobial
therapy (Remington & Klein, 1995).
The vast majority of infants admitted
to the NICU for suspected sepsis are
not infected but have symptoms
consistent with those of other medical
conditions that mimic sepsis, such as
hypoglycemia, delayed transition, or
transient tachypnea. Despite this fact,
these term infants are treated with
antibiotics for at least 48 h while
awaiting the results of the preliminary
blood culture report. However, even
blood culturing techniques can have
unacceptably low sensitivities (Jordan
& Durso, 2000). As microbiological
culture results are not usually available
until at least 48-72 hours after the
specimen reaches the laboratory, early
identification of infected cases is
recognized as a major diagnostic
problem in infants suspected to have
neonatal sepsis. In recent years,
haematological
and
biochemical
markers such as immature:total
neutrophil ratio, platelet count, Creactive protein (CRP), and various
cytokines have all been suggested as
being useful indicators for early
identification of septic infants (Ng et
al., 1997). Septic infections have been
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required to screen for multiple
organisms. Broad-range assays, based
on ribosomal genes (rDNA), are
designed to overcome this limitation.
Bacterial rDNA consists of highly
conserved nucleotide sequences that
are shared by all bacterial species,
interspersed with variable regions that
are genus- or species-specific. The
DNA sequences of the variable regions
form the basis of phylogenetic
classification of microbes (Doolittle,
1999).Numerous investigations also
have been carried out by use of
universal primers from the highly
conserved regions of the 16S rRNA
gene, which allows for amplification of
all bacterial species. Several recent
studies indicate that PCR may be
useful for detection of bacteria in
highly infected tissue specimens (e.g.,
resected heart valves or skin abscesses)
(Goldenberger et al., 1997 &
Rantakokko-Jalava et al., 2000). Lesscompelling
findings have
been
described with universal screening of
blood samples, most commonly due to
contaminant bacterial DNA (Ley, 1998
& Fredricks and Relman,1999). By
using PCR primers that are targeted at
conserved regions of rDNA, it is
possible to design broad-range PCRs
capable of detecting DNA from almost
any bacterial species. (Drancourt et al.,
2000; Janda and Abbott, 2002). Broadrange bacterial PCRs are more prone to
contamination with exogenous DNA
than other PCR assays and extra
precautions must be taken to ensure
that accurate results are obtained
(Millar et al., 2002). The PCR is the
most sensitive of the existing rapid
methods to detect microbial pathogens
in clinical specimens. In particular,
when specific pathogens that are
difficult to culture in vitro or require a
long cultivation period are expected to
be present in specimens, the diagnostic
value of PCR is known to be
significant (Yamamoto, 2002).
MATERIAL AND METHODS:
The study was done on 90
infants at NICU of Minia University
Hospital. All infants up to 1-month old
admitted to the NICU for sepsis
evaluation. The study included two
groups; cases group sixty infants who
were diagnosed clinically to have
neonatal sepsis and thirty infants
excluded to have sepsis at the clinical
level. Babies who had received
antibiotics before admission were
excluded. The clinical history was
taken as regards to gestational age and
clinical examination as regards to birth
weight, clinical symptoms in the form
of refusal of food, hypothermia,
lethargy, poor crying, diarrhea,
vomiting, fever, clinical signs in the
form of jaundice, pyoderma, cyanosis
abdominal
distention,
seizures
conjunctivitis, apnea, and tachypnea.
All blood samples were drawn by
physicians via venipuncture after
iodine preparation of the skin. Blood
volumes collected ranged from 2 to
3 mL; 0.5 to 1.0 mL was used for
blood cultures, 0.2 to 0.5 mL was used
in amplification of 16s rDNA gene and
the rest of the sample was centrifuged
and the serum was collected and used
for quantitation of the IFNγ, sCD14,
IL-6 and CRP.
Blood culture
0.5 to 1.0 ml of the drown
sample was aseptically inoculated to
blood culture bottles containing 10 ml
Tryptic Soy Broth (TSB) which is
capable of supporting growth of
common areobic, facultative and
anaerobic organisms from blood
specimens. All bottles were transported
to the laboratory as soon as possible
and immediately incubated at 37ºC in
an upright position. Subcultures were
taken at various incubation intervals.
After 24- to 48-h incubation, a small
quantity (0.1 to 0.5ml) of blood-broth
mixture were removed by means of a
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sterile syringe and needle and
subcultured according to the gram stain
result on blood, Chocolate and
MacConkey agar
incubated for up to 5 h at 37°C in room
air, with continuous shaking, after
which the cellular fraction of the
whole-blood sample was pelleted at
13,000 × g for 5 min at 4°C.
The agar plates were incubated
under aerobic conditions. However,
chocolate and blood agar plates were
incubated in a candle jar to facilitate
growth of Haemophilus influenza and
Neisseria and better growth of
Streptococci. Visible colonies were
identified after 24 hours of incubation
and a Gram stain using Preston
Murrell's modification method was
made. Standard biochemical tests were
performed on pure colonies of gramnegative isolates; Oxidase, indole, MRVP,
citrate,
motility,
Sugar
fermentation tests. For gram positive
isolates catalase, Coagulase, and
CAMP tests. Blood cultures, which
showed no visible growth and were
negative on Gram stain, subcultures
were done daily up to a maximum of
seven days before being discarded as
negative. Cytokine concentrations were
measured by a double-sandwich
ELISA technique using a commercial
kit specific for IL-6 (Cytimmune) IFN
γ and sCD14 (R&D Systems,
Minneapolis, MN). The detection limit
of the assay as indicated by the
manufacturer was 15.6 and 125 pg/mL,
respectively. Duplicate measurements
were performed for each sample.
Samples were diluted before analysis
as necessary. Dilution buffer was
provided by the manufacturer. Serum
CRP levels were determined by
turbimetric method using Turbitex
®CRP,Biocon®Diagnostic , Hecke 8,
34516 Vöhl/Marienhagen, Germany.
DNA extraction from whole blood
sample:
DNA
extraction
was
performed using Applichem DNA
extraction kit, 200 µl of total lysis
buffer and 20 µl of proteinase K
solution was added to each cell pellet
the whole sample was mixed by
inverting the tube and incubated at
37°C for 20 minutes. The sample was
intensively mixed for 20 seconds and
applied on a DNA purification
minicolumn GDI and centrifuged at
10000 - 15000 rpm for 1 minute the
minicolumn was placed with tube in
rack and added to which 500 µl of
wash solution AC1 then centrifuged at
10000 - 15000 rpm for 1 minute.The
minicolumn was transferred to a new 2
ml tube (supplied), added to which 400
µl of wash solution AC1, and
centrifuged at 10000 - 15000 rpm for 2
minutes. Dried minicolumn was
transferred to a new 1.5 ml tube and
add 100 or 200 µl of Tris buffer (10
mMTris · HCl pH 8.5), pre-warmed to
75°C. The sample was incubated at
room temperature for 5 minutes and
centrifuged at 10000 - 15000 rpm for 1
minute. The minicolumn was discarded
and purified DNA was stored at +4°C
for PCR.
Amplification of 16s RNA gene
PCR amplification and product
detection. Ten microliters of each
prepared specimen was added to 90 ml
of a PCR master mix consisting of
0.05units/µl Taq DNA Polymerase
(recombinant) in reaction buffer, 4mM
of MgCl2 and dNTPs (dATP, dCTP,
dGTP, dTTP)
0.4 mM of each
(Fermentas), and either a 25 mM
concentration of each of forward
primers, 5'-AAC TGG AGG AAG
Whole-blood specimen preparation
for PCR analysis.
Pre PCR enrichment: 200 to 500 μl
of whole blood was added to 4 ml of
tryptic soy broth (TSB) (Difco
Laboratories, Detroit, Mich.) and
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GTG GGG AT3'; reverse primer, 5'AGG AGG TGA TCC AAC CGC A-3'
. This primer can amplify 380 bp of
highly conserved sequence of bacterial
16s rDNA gene (Jordan and Durso,
2005). Cycling conditions included a 2minute denaturation step at 95°C,
followed by 35 cycles of 30 seconds at
95°C, 60 seconds at 60°C, and 30
seconds at 72°C.
RESULTS :
Table: 1 Clinical symptoms of septic group and control groups.
Refusal of food
Hypothermia
Lethargy
Poor crying
Diarrhea
Vomiting
Fever
Excessive crying
Case No. 60
(%)
37(61.7%)
12(20%)
20(33.3%)
15(25%)
4(6.6)
5(8.3%)
6 (6.6%)
3(5%)
Control N.
30(%)
4(13%)
3 (10 %)
4 (13 %)
3 (10 %)
1 (3.3 %)
2 (6.6 %)
1 (3.3 %)
1(3.3%)
From the evaluated cases the
refusal of food was the symptom of the
highest significance P< 0.001 followed
by fever
P value
0.0001**
0.186
0.044*
0.049*
0.559
0.534
0.505
1
P= 0.024 then lethargy and poor
crying. The other symptoms where non
significantly different between cases
and controls.
Table (2): Sensitivities, specificities, positive prdective and negative predictive
values of the clinical symptoms.
Refusal of food
Hypothermia
Lethargy
Poor crying
Diarrhea
Vomiting
Fever
Excessive crying
Sensitivity
61.66%
20%
33.3%
25%
6.6%
18.6%
21.7%
5%
Specificity
86.7%
90%
86.7
90
90
75
96.7
86.7
As shown in the table (2) all the
symptoms show low sensitivities with
the refusal of food has the highest one
(61.66%) while excessive crying was the
least sensitive symptom (5%). At the
same time specificities of the clinical
was somewhat acceptable with the
highest was that of fever(96.7%) the
5
PPV
90.24
80
83.33
83.33
80
61.1
92.9
66.7
NPV
53.06
36
39.4
37.5
13.84
30.4
38.15
33.3
least specific symptom was vomiting.
The over all positive predictive values of
the different clinical signs were high
with the fever at the top (92.9%)
followed by refusal of food (90.24%)
however, the negative predictive values
were low.
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Table (3): Clinical signs of septic vs. control groups
Jaundice
Pyoderma
Cyanosis
Abdominal distention
Seizures
Conjunctivitis
Apnea
Tachypnea
Poor capillary refill
Bradycardia
Case
N.(60)
19(31%)
7(11.7%)
15(25%)
6(10%)
5(8.3%)
4(6.6%)
8(5%)
3(5%)
2(3.3%)
7(11.7%)
From the evaluated cases and
control, all the clinical signs were
non significantly different between
case and control groups however the
Control
N.(30)
11(36%)
……
4(13.3%)
2(6.6%)
2(6.6%)
1(3.3%)
2(6.6%)
1(3.3%)
1(3.3%)
1(3.3%)
P value
0.32
0.00**
0.201
0.646
0.823
0.515
0.718
0.718
0.469
0.001**
most prevalent clinical sign was
neonatal jaundice (31%) followed by
cyanosis (25%) and tachypnea.
Table (4): Sensitivities, specificities, positive and negative predictive values of the
clinical signs.
Jaundice
Pyoderma
Cyanosis
Abdominal distention
Seizures
Conjunctivitis
Apnea
Tachypnea
Poor capillary refill
Bradycardia
S.
31.7
6.6
25
10
8.3
6.7
13.3
15
3.3
11.3
As shown in the table (4) all the
clinical signs show low sensitivities
with jaundice have the highest one
(31.7%) while poor capillary refill
been the least sensitive symptom
(3.3%). At the same time specificities
of the clinical were significantly high
with the highest was that of
pyoderma (100%) the least specific
Sp.
70
100
86.7
86.7
93.3
96.6
93.3
93.3
96.7
93.3
PPV
67.9
100
78.9
60
71.4
80
80
81.8
66.7
77.8
NPV
33.9
34.9
36.6
32.5
33.7
34.1
35
34.4
33.3
32.5
sign was jaundice 70%. The over all
positive predictive values of the
different clinical signs were high
with the pyoderma is the most
specific (100%) followed by
tachypnea (81.8%) however; the
negative predictive values were
apparently low.
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Table (5): Blood culture results from cases and control groups.
Case
Cont
N
32
2
Positive
%
53.33
6.67
Negative
n
%
28
46.67
28
93.33
P
0.0
This table shows culture
positivity rate, there was 32 positive
blood cultures while the control show
Sensitivity
specificity
53.33
93.33
two positive cultures. The culture
was highly specific (93.3%) but
moderately sensitive 53.3%.
Table (6): The isolated microorgansms from blood sample.
Microorganism
Cases
Controls
Coagulase negative staphylococci
10(31.25%)
2
S. aureus
GbS
Listeria
9(28.1%)
1((3.1%)
1(3.1%)
Klibsiella
Pseudomonas
E.coli
2(6.25%)
2(6.25%)
7(21.9%)
Of a total of 60 blood cultures
from clinically suspected sepsis, 32
were positive (positivity rate of 54%)
gram positive organisms were the most
common. The most common causative
organisms were staph coagolase
negative (31.25%) Staphylococous
aurous (21.9%) Klebsiella (6.25%) and
GBS (3.1%) Pseudomonas (3.1%)
Escherichia coli (28.1%). The two
positive cultures from the control
group revealed CoNS as the only
isolated organism
Table (7): Serum concentrations of inflammatory mediators at the day 0 of clinical
diagnosis of sepsis
Cd14
IFN γ
IL-6
CRP
Cases n.20
0.415 (0.33- 0.69)
71
(24-460)
1521.5 (50-2250)
15 (5–89)
7
Controls
0.28(0.20-0.35)
9 (2-18)
41 (15-62)
6 (5–14)
p
0.001*
0.001*
0.0001**
0.001
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Table (8): Sensitivities, specificities, positive prdective and negative predictive
values of the estimated mediators.
IL-6 > 60 pg/ml
CRP >10 mg/L
CD14 > 0.35 ug/ml
IL-6 >60pg/ml and/or
CRP>10 mg/L
S.
91.7
82
88.3
SP.
80
73
96.66
PPV
90.16
86
98.1
NPV
82.75
76
80.6
96.7
93.3
96.7
93.3
The immunological mediators show high sensitivities with IL-6 has the
highest one (91.7%) while followed by sCD14 (88.3%) then CRP (82%) while the
combined sensitivity of CRP & IL-6 was much higher than any other.
Table (9): Differencies between levels of the estimated mediators in infants with
gram positive sepsis and those with gram negative srpsis
sCD14
IFN γ
IL-6
CRP
G +ve sepsis
0.38 (0.33-0.55)
75 (45-320)
900(170 -1800)
G-ve sepsis
0.55 (0.42 -0.86)
270 (35 - 460)
1950(1520-2200)
p
0.01
0.001
0.000
15 (5-30)
45 (16-71)
0.0001
There was significantly higher
CRP, IFNγ, IL-6, and Cd14 serum
level in patients with Gram-negative
sepsis, than that in infants the patient
with gram positive sepsis, P≤0.002.
Table (10): The result of PCR ampilification of the 16s rRNA gene from whole blood
samples of case and controle group
Case
Cont
Positive
No.
%
56
93.3
3
10
Negative
No.
%
4
6.67
27
93.33
P
0.0
From the 60 cases, 56 were
PCR positive for 16s rRNA gene with
sensitivity 93.3% specificity 90%, PPD
95% and NPD 85%. One of the four
Sensitivity
93.33
specificity
90
PCR negative was culture positive.
Three samples of the control group
were PCR positive
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Figure (1): Agarose gel electrophoresis of PCR product.
The picture shows the amplified sequence of 16s rRNA gene 370-380 bp.
DISCUSSION:
About five million neonatal
deaths occur worldwide every year,
98% of which occur in developing
countries, particularly Asia and Africa.
Infections such as tetanus, pneumonia,
septicemia, meningitis, and diarrhoea
account for 30–50% of neonatal deaths
in developing countries (Darmstadt,
2001). Infections in the neonate are
most important cause of mortality and
hospitalizations in the neonatal
practice. Early recognition of sepsis in
neonates is difficult. Early diagnosis
and timely treatment of neonatal
infections is essential (Yadav et al.,
2005). The diagnosis of sepsis is
difficult in neonates admitted to the
NICU due to nonspecific clinical signs.
Therefore, reliable indicators of sepsis
would be helpful in an accurate
diagnosis, resulting in decreased
unnecessary use of antibiotics.In the
current study the clinical manifestaions
were studied carfuly but unfortuantly
niether of them can conclud presence
or absense of sepsis, even the most
specific signes, were of very low
sensitivities. Of these features refusal
of feeding followed by tempretur
instability in the form of fever or
hypothermia, and lethergy were the
most significant these results were
comparable to those of (Karthikeyan
and
Premkumar,
2001).
These
symptoms, however, although having
good specificities & PPVs, they lake
reliable sensitities & NPVs ie. they are
not good at detecing the disease and
their absence is not good at exclusion
of sepsis. An other reason lowering the
reliablity of clinical features in giving a
definit diagnosis, is the varable
predominance of these findings along
a considrable number of studies
Makhoul et al., 2006; Oray- Schrom et
al., 2003; Gonzalez et al., 2003;
Fanaroff et al., 1998. As previously
reviewed, the "gold standard" for
diagnosing neonatal sepsis remains the
blood culture, even though, in many
cases, blood cultures are negative in the
face of strong clinical indicators of
septicemia and even in autopsy-proven
disseminated bacterial or fungal
infection. In the current study, blood
culture positivity rate in neonatal
septicemia cases was 54%, whereas in
46% of cases there was no growth.
Similar positiviy rate were obtained by
Aftab and Iqbal, 2006. A relatively
higher culture positivity rate has been
reported by Rahman et al., 2002 where
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positivity rate of blood culture was
62.8%. However, a low blood culture
positivity rate (9.5%) has been
observed by Borna et al., 2004, (20%)
by kapoor et al., 2005. This relatively
moderate culture positivity rate in our
study might be due to assurance that
the infants did not receive antibiotics
before sampling, was one of the factors
improving the positivity rate of culture.
On the other hand, administration of
intrapartum antibiotics is routinely in
our hospital cannot be neglected. In
addition, the relatively small sample
volume used in culture may reduce the
opportunity of microbial growth. These
variable rates of positivity enhance the
need for more accurate and reliable
parameter for sepsis diagnosis, even
positive cultures, are susceptible for
contamination. Pourcyrous and coworkers, 1993 recognized that 83% of
blood cultures yielding organisms with
low-grade or questionable virulence
were the result of contamination during
collection especially when using
broken needle technique. One more
recent study done upon
Infants
younger than 60 days, admitted for
severe pneumonia or suspected
sepsis/meningitis were prospectively
evaluated using complete blood
culture, in Manila, Philippines; the
study revealed that gram-negative
enteric bacteria are the predominant
causes
of
community-acquired
infections in Filipino infants below 2
months old, and more specifically
those with early onset sepsis.
pathogen as the causative organism of
sepsis and enhances the need of every
centre to specify the commonest
pathogen causing sepsis in its NICU.
CoNS are the leading cause of
bacteremia in the NICU setting, where
immunologically immature infants rely
on invasive devices for their care.
Venous catheters have been implicated
in more than one-half of the cases of
CoNS bacteremia in NICUs (Kerur et
al., 2006). The present study also
assures that emergence of CoNS as an
important cause of neonatal sepsis, in
spite of being in milder forms and
better outcomes than that caused by
Gram-negative
bacteria;
similar
findings were presented by Makhoul et
al., 2006. CoNS also were the single
bacterium isolated from control
samples meaning that it can be
considered the commonest cause of
blood culture contamination. The
second most important pathogen
isolated in our study was staphylococcus aureus, this finding also was
reported by Ronnestad and coworkers,
1998 who found that CoNS was the
commonest causative organism of
sepsis at all studied groups; very
early, early and late sepsis, followed by
staphylococcus aureus which was the
commonest between the early onset
group. Likewise, Anwer and his team,
2000, reported that CoNS and S.
aureus were the commonest isolates.
Another study conducted by Yalaz and
his team, where S. Aureus accounts
for (13%) of septic neonates and comes
second to CoNS, 72% of isolated S.
aureus were methecillin resistant.
Meanwhile Aftab and Iqbal, 2006,
found
it
the
most
frequent
(32%).Considering the Gram-negative
organisms, inspite of being less
frequent than gram positive bacteria,
remain important group of causing
sepsis especially E.coli . In contrary to
our data E. coli was the commonest
isolated pathogen in multiple studies.
(Quiambao et al., 2007).In the
present study Gram positive cocci
where the commonest isolated
pathogens with CoNS was the
commonest of them, these data was
comparable to that of Sarkar et al.,
2006 & Park et al., 2007; Aftab and
Iqbal,
2006.
However,
theses
differences in the microbial prevalence
make it difficult to generalize a definite
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EL-MINIA MED., BULL., VOL. 18, NO. 2, JUNE, 2007
Shokry et al
_______________________________________________________________________________
(Aftab and Iqbal, 2006; Ojukwu et al.,
2006).
evaluated serially (Gonzalez et al.,
2003). However another study found
that there is no benefit from serial
evaluations
and
the
initial
measurement is enough (Magudumana
et al., 2000). Also in the present study,
IL-6 was significantly higher levels in
septic group median 1521 pg/ml (502250), compared with non septic
neonates 41 pg/ml (15-62), these
results were comparable to that of
Martin et al., 2001 where median
serum levels in newborn infants (n =
32; gestational age 39 ± 3 weeks) with
sepsis: 1620 pg/mL; in nonseptic
neonates: 42 pg/mL these data
recommends the use of IL-6 together
with CRP as reliable marker of sepsis
Romagnoli et al., 2001, found higher
range of interleukin 6 (487-10000
pg/ml) but the median was running
with the present data. Gonzalez et al.,
2003, in spite of giving the same
significant difference, surprisingly,
giving much more lower values than
obtained at our and the former studies
median (40 vs. 13).At the present
study, the diagnostic value of serum
IL-6 level was higher than that of the
other estimated mediators with a cutoff value 60 pg/ml the sensitivity
91.7% and specificity 80%, positive
and negative predictive values were
90.16 %& 82.75% respectively, this
makes IL-6 is a superior on CRP as a
diagnostic marker for sepsis, similar
finding obtained by, Ceccon et al.,
2006 who reported that sensitivity of
serum IL-6 88.9%, 80 %, 76.2%, 90.9.
Rite Gracia et al., 2003 found higher
sensitivity of IL-6 was 100%. Two
previous studies reporting elevated
levels of CD14 in serum of neonates
with sepsis (Blanco et al., 1995 &
Berner et al., 2002). In the study done
by Blanco and his collaegues,
however, the measurement of sCD14
was performed at a postnatal age of
about 2 weeks, with a wide time range.
Berner and his team, 2002 found
However, these data are
contradicted by other studies that
consider klebsiella Pneumonia is the
most frequently isolated pathogen from
blood of septic neonates; Bell et al.,
2005, reported Klebsiella as the
commonest pathogen, with the highest
mortality, other studies by Kapoor et
al., 2005.While some studies reported
the capability of newborn infants to
produce inflammatory cytokines has
been considered immature (Pillay et
al., 1994 & Rowen et al., 1995),
Schultz and his team, 2002 refuted the
view that the inflammatory response in
preterm infants is immature and on
contrary, they assured that an enhanced
production
of
proinflammatory
cytokines could be demonstrated both
spontaneously and after endotoxin
challenge directly at the cell level in
term and preterm infants. Likewise,our
data
showed
this
enhanced
inflammatory response in the form of
elevated levels of IFN-γ and sCD14,
CRP and proinflammatory response in
the form of elevated levels of IL-6 in
septic neonates compared with healthy
ones.Early in the course of neonatal
sepsis, IL-6 was produced rapidly and
peaked on day 0, but its half-life was
short and could fall back to its baseline
value within 24 hours. This specific
property of IL-6 rendered it useful as a
very early alarm hormone, but because
of its short half-life, clinicians could
not rely on this marker alone for the
diagnosis of infection, because in most
circumstances it was uncertain at which
stage of infection blood was taken for
IL-6 determination. As the purpose of
the current study was the assessment of
the role of mediators in the diagnosis
of neonatal sepsis, we were satisfied by
single measurement at time of clinical
suspicion. In its use as a following up
marker, IL-6 was recommended to be
11
EL-MINIA MED., BULL., VOL. 18, NO. 2, JUNE, 2007
Shokry et al
_______________________________________________________________________________
significantly elevated levels of sCD14
and LBP in samples from septic
neonates collected at or immediately
after birth when compared to samples
from healthy control neonates. These
findings indicate that the neonate at the
moment of birth is capable of releasing
relevant amounts of sCD14 and LBP,
but it is also important to mention that
such an excessive response, as in
adults, with sepsis is lacking (Froon et
al., 1995).In the present study sCd14
serum levels were also significantly
higher in septic group than those of
control group, this supports the opinion
of Bas et al., 2004 who has reported
that the soluble form behaves as an
acute phase protein. It was supposed
that the soluble form of CD14 can
compete with the membrane-bound
form for LPS binding and thus can
reduce the effects of endotoxin (Haziot
et al., 1995). The elevated concentrations of sCD14 also documented for
the 19 newborns with gram-positive
infections. These data show that grampositive bacteria, such as S. aureus,
which do not possess LPS as
constituents of their cell wall, are
capable of inducing a significant
secretion of CD14 in vivo. It has been
described recently that both LPS and
cell wall preparations of S. agalactiae
induce TNF- secretion from human
monocytes in a CD14-dependent
manner (Cuzzola et al., 2000).
Comparison between gram-positive
sepsis and gram-negative sepsis
revealed significantly higher level of
sCD14 with subsequent higher levels
of other mediators in patients with
Gram-negative sepsis indicating that
LPS is more powerful inducer of
sCD14 and the following mediators
than peptidoglycan. Berner et al., 2002
have extensively studied the kinetics of
sCD 14 along the course of sepsis, but
he did not compare the results of gram
positive and Gram-negative induced
sepsis. The present study revealed
significantly elevated serum concentrations of the assayed immunological
mediators in neonates with Gramnegative sepsis than those with grampositive sepsis. This finding can help
in understanding the more sever forms
of sepsis, and the higher morality rates
occurring with Gram-negative than
gram-positive sepsis. Some other
reports suggested that coagulasenegative infections might probably
cause a weaker inflammatory response
than
infections
with
other
microorganisms (Franz et al., 1999,
Laborada et al., 2003). However,
Verboon-Maciolek, 2006, and his team
reported that no significant difference
between cytokine level produced by
CoNS and that induced by the more
virulent S. aureus. Standard diagnosis
of systemic bacterial infection depends
on growth in culture, which requires at
least 12 to 72 h for detection. The most
rapid tests are latex agglutination tests
and Gram stains, which are less
sensitive than culture and molecular
methods (Goldenberger et al., 1997).
Molecular biological methods for
detection of nucleic acids have been
shown to have greater sensitivity than
immunological and staining methods.
The use of PCR primers that target
DNA regions that are conserved in
bacteria for the purposes of DNA
sequencing and detection of bacteremia
has been described (Klausegger et al.,
1999). There are numerous examples
of PCR-based assays for detecting
bacteria in blood, including Streptococcus pneumoniae DNA from whole
blood or inoculated Peds Plus bottles
(Friedland et al., 1994) and coagulasenegative Staphylococcus sp. from
blood culture bottles (Carroll et al.,
1996). In the present study, PCR assay
of 16s rRNA gene was found to be of
high sensitivity, specificity, PPD and
NPD (93.3%, 90%, 95%, 85%,
respectively). Similar findings were
obtained by Jordan, 2000. In that,
12
EL-MINIA MED., BULL., VOL. 18, NO. 2, JUNE, 2007
Shokry et al
_______________________________________________________________________________
study the primer used was the same to
that used by us and was capable of
amplifying a highly conserved
sequence of broad range of bacteria. In
contrast to the present study Makhoul
et al., 2006, found lower sensitivity for
PCR usage in the diagnosis of neonatal
staphylococcal bacteremias, and he
reasoned that by the probable low
bacterial load of the samples and the
lack of pre PCR enrichment. Despite
all
efforts
taken
to
avoid
contamination, the risk is still higher
for broad-range PCR than for assays
that are more specific. Using a
sensitive and rapid method combining
broad-range PCR amplification of
bacterial 16S rDNA fragments, Grahn
et al. 2003, found contaminating
bacterial DNA in reagents used for
PCR reactions, further identified as
water-borne bacteria. In the present
study, there were 4 PCR positive
samples in the control groups
indicating the possible contamination
of samples during PCR assay, while,
presence of culture positive PCRnegative samples might be reasoned by
blood culture contamination. A
multiplex approach was developed to
detect neonatal sepsis, coamplifying
portions of the 16S rRNA gene along
with the housekeeping gene for
GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) (Laforgia et al., 1997).
In that study, among the 33 newborn
infants classified as being at risk for
early-onset sepsis, Laforgia et al. were
able to detect the 16S rRNA gene by
PCR in all four of the culture-proven
sepsis cases, as well as in two samples
with negative culture results. Finally, a
PCR assay using primers, which
recognize an 861-bp fragment of the
16S rRNA gene, was suggested for use
in triaging bacterial sepsis (McCabe et
al., 1995). That study revealed the
successful amplification of the rRNA
gene from 12 different species of
bacteria, including gram-negative and
gram-positive organisms, without
amplifying human genomic DNA.
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‫‪Shokry et al‬‬
‫_______________________________________________________________________________‬
‫تقييم قياس الـ ‪ 16s rDNA‬بتفاعل البلمرة المتسلسل )‪ (PCR‬وبعض الوسائط‬
‫المناعية مقارنة بمزارع الدم فى تشخيص التلوث الدموى‬
‫الجرثومى فى األطفال حديثى الوالدة‬
‫محمود شكرى محمود* – محمد إبراهيم بسيونى* – سحر أبو العيون* –‬
‫محى معاذ* – سمير تامر**‬
‫أقسام *الميكروبيولوجى والمناعة و **األطفال – كلية طب المنيا‬
‫يعتبر التلوث الدموي الجرثومي من أهم األمراض التي تصيب األطفال الحدديثي الدوةدو وكدذل‬
‫يعتبر من أهم اسباب الوفاو في الألطفال المبتسرين‪ .‬وقد هدفت هذه الدراسدة إلدى التعدرع علدى‬
‫الميكروبات التدي تسدبب التلدوث الددموي الجرثدومي فدي األطفدال حدديثي الدوةدو ومعرفدة أنمداط‬
‫مقاومتها للمضادات الحيوية وكذل تقييم دور الوسائط المناعيدة المتتلفدة مثدل السديتوكينات فدي‬
‫التشتيص المبكر للمرض وهدفت كذل إلدى تقيديم تشدتيص التسدمم الددموي الجرثدومي بطريقدة‬
‫تفاعل البلمرو المتسلسل ومقارنتها بالطريقة التقليدية وهي مزرعة الدم‪.‬‬
‫وقد شملت الدراسة مجموعتين من األطفال‪ :‬المجموعة األولى تتكون من (‪ )60‬طفالً يعانون من‬
‫التلوث الدموي الجرثومي و المجموعة الثانيدة تتكدون مدن (‪ ) 00‬طفدال اسدتبعد أن يكدون عنددهم‬
‫هذا المرض‪.‬‬
‫وقددد تددم جمددن العينددات مددن وحدددو المبتسددرين بالمستشددفى الجددامعي ونقلددت إلددي معمددل قسددم‬
‫الميكروبيولوجي والمناعة بالكلية ثم أجريت لها الفحوص التالية‪:‬‬
‫‪ )1‬مزرعددة للدددم والعددزل والتعددرع علددي الميكروبددات المسددببة وكددذل أنمدداط مقاومتهددا‬
‫للمضادات الحيوية المتتلفة‪.‬‬
‫‪ )2‬معايرو انترليوكين‪ , 6-‬وإنترفيرون جاما ‪ ,‬وسدي دي ‪ 11‬الدذائب ‪ ,‬بطريقدة ايليدزا‪ ,‬أمدا‬
‫بروتين ‪ C‬المتفاعل فقد تمت معايرته بطريقة التيربمتري‪.‬‬
‫‪ )0‬تكبير الجين التاص بالـ (‪ )16s rDNA‬والموجود حصرا في البكتريا من عينات الدم‪.‬‬
‫وقددد أرهددرت النتددائز أن مزرعددة الدددم أقددل كفائددة فددي تشددتيص المددرض مقارنددة بتفاع دل البلمددرو‬
‫المتسلسددل ‪ % 30, %3030‬علددي التددوالي‪ .‬وقددد أرهددرت نتددائز مزرعددة الدددم أن الميكددروب‬
‫العنقددودي إبيدددرمي هددو األكثددر شدديوعا بددين الميكروبددات المعزولددة يليدده الميكددروب العنقددودي‬
‫أوري ثم ميكروب اييشريشيا كوةي (‪ %2133 ,%2.31 ,%01323‬علي التوالي)‪.‬‬
‫وقددد أرهددرت النتددائز أيض د ا أهميددة قيددا الوسددائط المناعيددة وأهمهددا انترليددوكين ‪ 6‬حيددث كددان ذا‬
‫حساسددية أعلددى فددي تشددتيص المددرض مددن حساسددية بددروتين ‪ C‬المتفاعددل ‪ %3139‬مقارنددة ب‬
‫‪ %.2‬علي التوالي ولكن قياسهما معا له حساسية أعلي من قيا كل منهما على حدو ‪.%3639‬‬
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