Content uploaded by Mervat Elanany
Author content
All content in this area was uploaded by Mervat Elanany on Nov 10, 2017
Content may be subject to copyright.
Med. J. Cairo Univ., Vol. 77, No. 3, June: 291-300, 2009
www.medicaljournalofcairouniversity.com
Early diagnosis of Neonatal Sepsis: The Role of 16S rRNA Gene
Sequence Analysis
ISMAIL M. EL-HAWARY, M.D.*; NADA N. NAWAR, M.D.**; MERVAT G. EL-ANANY, M.D.**;
MARIAM A. YONAN, M.D.** and MANAL EL-SEWEIFY, M.D.***
The Department of Pediatrics, Cairo University* and the Department of Clinical Pathology, Cairo** & Fayoum*** Universities.
Abstract
Background:
Early diagnosis and treatment of newborn
infants with suspected sepsis are required to reduce sepsis
associated mortality and to prevent severe life- threatening
complications. Clinical diagnosis is difficult and, blood culture,
is often negative in the face of strong clinical indicators of
septicemia, on the other hand, molecular diagnostics have
proved to be a valuable adjunct for detection of neonatal
bacteremia. The purpose of this study was to evaluate the role
of 16S rRNA gene sequencing for rapid and sensitive diagnosis
of clinically septic neonates with pathogen identification to
allow for early and specific treatment of neonatal infections.
Subjects and Methods: A prospective study conducted in
both NICUs of Kasr Al Aini hospital and Children’s hospital,
Cairo University over a period of 3 months from April 2008
to June 2008. Fifty neonates undergoing sepsis evaluation
were included in this study. Risk factors, clinical manifestations
and hematological findings suggestive of sepsis were recorded.
Bloodculture and broad range 16S rRNA polymerase chain
reaction (PCR) were collected from clinically septic neonates
to confirm diagnosis of sepsis. Genotypic identification of
bacteria by sequence analysis of the 16S rRNA gene was
performed on all samples positive in PCR.
Results:
Fifty neonates suspected clinically of having
sepsis were included in the study. 61 samples were collected
as a part of evaluation of infection, 50 samples upon admission
and 11 samples as follow-up due to persistence of clinical
manifestations. The rate of culture proven sepsis was 19.7%
(12/61). With the molecular method of broad range 16S rRNA
PCR, the detection of bacteria improved to 29.5% (18/61).
PCR revealed sensitivity, specificity, positive predictive value
and negative predictive value of 91.7%, 85.4%. 61% and
97.6% respectively, while the accuracy of this test was 86.7%.
So, compared to culture, the 16S rRNA PCR demonstrated a
high negative predictive value for ruling out neonatal sepsis.
According to the PCR results, hyperthermia, feeding intoler-
ance and abdominal distension were found to be significant
among neonates with positive PCR. As for hematological
manifestations, we found a significant association between
increasing number of abnormal hematological findings and
PCR positivity (
p
=0.006,
p
for ordinal correlation =0.01).
Odds ratio for having 2 or more abnormal hematological
findings and PCR positivity was 3.77 (95% CI=1.2-12.3).
Sequence analysis was done for further identification of
organism on all samples positive in PCR. The sequenced PCR
was in accordance with blood culture results in 10 samples
while one sample showed conflicting results (91 % agreement),
Seven samples showed no growth by culture but were identified
by sequence analysis. The sequencing-based identification of
these isolates included: Enterobacter cloacae, Staphylococcus
carnosus, Proteus mirabilis, Haemophilus influenzae, Strep-
tococcus pyogenes and Klebsiella sp. One sample yielded
insufficient DNA to be analyzed.
Conclusion:
Broad range 16S rRNA PCR can be used to
rule out sepsis. Both PCR and sequence analysis can provide
additional diagnostic data that cannot be obtained with the
use of broad-range PCR or routine laboratory tests alone. In
neonates with clinically suspected sepsis, a primary decision
could be made when the PCR results are completed, and
within few hours, genotypic identification from the sequencing
could be available to allow for early treatment.
Key Words:
Neonatal sepsis – PCR – Sequence analysis.
Introduction
NEONATAL
sepsis is one of the most common
reasons for admission to neonatal units in devel-
oping countries. It is a major cause of mortality in
both developed and developing countries
[1,2]
.
Early diagnosis and treatment of newborn infants
with suspected sepsis are required to reduce sepsis
associated mortality and to prevent severe life-
threatening complications. Unfortunately, diagnosis
is difficult, symptoms and signs are usually non
specific and laboratory methods usually lack both
sensitivity and specifity. As a result, a considerable
number of newborns are treated with antibiotics
for suspected neonatal sepsis even though not
infected
[3]
.
The "gold standard" for diagnosing neonatal
sepsis is the blood culture. Whereas a positive
culture confirms the diagnosis of sepsis, a negative
291
292
Early diagnosis of Neonatal Sepsis: The Role of 16S rRNA
culture does not exclude sepsis. In many cases,
blood cultures are negative in the face of strong
clinical indicators of septicemia. Some of the
sepsis-like episodes with negative blood cultures
may be caused by true infection with a false-
negative blood culture, while others may be initiated
by factors such as respiratory or gastrointestinal
diseases
[4]
.
Maternal antibiotics given in preterm
deliveries or insufficient sample size may cause
the blood cultures to be falsely negative for com-
mon neonatal pathogens. Furthermore, results of
the culture are not available before 48-72 hours
[3,5]
.
Early diagnosis of neonatal sepsis and sepsis-
like illness is an important goal, and new techniques
to identify causative organisms are required. Mo-
lecular diagnostics have proved to be a valuable
adjunct for detection of neonatal bacteremia. De-
tection of bacterial DNA in the blood has been
accomplished by broad-range polymerase chain
reaction (PCR) amplification of highly conserved
regions of the 16S ribosomal RNA, a gene univer-
sally present in bacteria but absent in humans.
Detection of as few as 10 organisms per ml of
whole blood has been reported 6 and detection of
bacterial DNA in blood samples of neonates rep-
resented a rapid and sensitive supplement to blood
culture in diagnosing bacterial sepsis in neonates
[7,8,9]
.
Moreover, genotypic identification of microor-
ganisms by 16S rRNA gene sequencing has
emerged as a more objective, accurate, and reliable
method for bacterial identification, with the added
capability of defining taxonomical relationships
among bacteria. DNA sequencing of conserved
regions within phylogenetically informative genetic
targets, such as 16S rRNA gene, is promising as
a means for identifying bacteria
[10,11,12]
.
In the cases with negative blood culture and
positive PCR, DNA sequencing is of great benefit,
as the PCR sequencing can detect a pathogen that
was not identified by conventional blood culture.
In these cases a primary diagnosis can be made
and within few hours, or even before the next
antibiotic dose, additional data from the sequencing
could be available
[3]
.
Aim of work:
The study targeted to determine
whether the use of 16S rRNA gene sequencing to
screen blood for bacteria, will be a useful test for
rapid and accurate identification of a causative
organism in clinically septic neonates that would
modify antibiotic therapy. We sought the possible
applicability of DNA sequencing in the clinical
settings and to interpet the false-positive and false-
negative PCR results.
Materials and Methods
Patient selection:
Fifty neonates undergoing sepsis evaluation in
either NICUs of Kasr Al Aini hospital or Children’s
hospital, Cairo University over a period of 3
months, from April 2008 -June 2008, were included
in this prospective study. Infants were suspected
of having sepsis on admission or developed clinical
manifestations suggestive of infection during their
hospital stay.
Baseline data for studied infants including
gender, birth weight, gestational age, mode of
delivery and cause of admission, were documented.
Suspected sepsis was defined as any clinical
concern for infection that required the start of
intravenous antibiotics before laboratory or micro-
biological evidence of infection. Clinical manifes-
tation suggestive of sepsis included the presence
of two or more of the following clinical criteria:
respiratory distress or tachypnea (respiratory rate
of >60 breaths per minute), apnea (cessation of
respiration for >20 seconds, occurring at a rate of
>2 times per hour or any single episode requiring
positive pressure ventilation), tachycardia (heart
rate > 160 beat per minute that, bradycardia (heart
rate of <100 beats per minute), decreased perfusion
(capillary refill of >3 seconds or cold extremities);
hypothermia (rectal temperature of <36
°
C), hyper-
thermia (rectal temperature of >38
°
C), feeding
intolerance (increased gastric residuals of >30%
of food volume in more than 2 feeds within 24
hours), abdominal distension, seizures, irritability,
lethargy, or decreased activity. These clinical fea-
tures were found to be strongly suggestive of
infection
[13]
.
Factors associated with increased risk for in-
fection were also identified such as prolonged
rupture of the membranes (>18 hours), mechanical
ventilation, total parenteral nutrtion (TPN), umbil-
ical catheterization and prolonged length of hospi-
talization.
Laboratory investigations were done for these
infants including total white blood cell count
(<5000 or >20000 cells per mm
3
), immature-to-
total neutrophil ratio (I:T ratio
≥
0.2) and platelet
count (<150000 cells per mm
3
). Two or more
abnormal parameters were considered as helpful
Ismail M. El-Hawary, et al.
293
for diagnosis of infection
[14,15]
. Blood culture
was collected upon suspicion of sepsis to detect
the presence of pathogens and their antibiotic
sensitiviy. Follow-up of these patients was done
to record total length of stay and outcome.
Clinical specimens:
Between 1.0 and 2.0 ml of
whole blood samples were drawn for culture using
blood culture bottles (Peds Plus; Becton Dickinson).
No additional blood was collected from the infants
for the purposes of molecular analyses. Aliquots
of broth from blood culture bottles after 4-6 hours
of incubation were collected and saved at -70
°
C.
61 samples were taken from our patients, 50 sam-
ples upon admission due to suspected sepsis and
11 samples as follow-up due to persistence of
clinical manifestations suggestive of infection.
Blood culture processing:
An automated con-
tinuous-monitoring blood culture system, BACTEC
9120 (Becton Dickinson, Sparks, MD.) was used.
Bottles were incubated for 5 days before discarding
as negative and were sub-cultured according to the
laboratory operating procedure if they flagged
before this time. Identification tests were done
according to standards
[16]
.
Molecular analysis:
DNA extraction and PCR amplification:
Extraction: For the 61 aliquets of broth stored
at -70
°
C, nucleic acids were extracted by using
QIAGEN (R) RNA extraction kit (Q IAGEN (R),
Germany). Qiagen Kit according to the manufac-
turer’s instructions.
Primers:
The following pirmers were synthe-
sized at Metabion Company, Germany:
Forward: 536f 5" CAGCCGCGGTAATAC,
Reverse: RP2 5" ACGGCTACCTTACGACTT
Amplification: A 50
µ
l PCR mixture contained
the following: 100 mM Tris-HCL (pH 8.3); 200
µ
M (each) dATP, dCTP, dGTP and dTTP; 0.150
µ
M (each) primer; 1.5
µ
l of Ampli Taq; and 5 mM
MgCl
2
, genomic DNA at concentration of 200
ng/
µ
l and double distilled water up to 50
µ
l final
volume. Amplifications were performed on thermal
cycler, PTC100 Mil Research, USA. A total of 39
cycles were performed, and each cycle consisted
of 30 s of melting at 95
°
C, 30 s of annealing at
60
°
C, and 45 s of extension at 72
°
C. Prior to the
first cycle, the samples were heated to 95
°
C for
10 min, and the last cycle was followed by a final
extension at 72
°
C for 10 min. From final PCR
products, 10
µ
l were mixed with 2
µ
l gel loading
buffer 6X stock, loaded onto a 1.5% agarose gel
containing ethedium bromide at concentration of
0.5
µ
g/ml.
Visualisation:
The gel was subjected to electro-
phoresis in 1X TAE buffer for a suitable time that
allows the bromophenol blue to run 2/3 of the gel
length at 120 volt. A 100 bp DNA ladder (Gibco
BRL, life Technologies, Gent, Belgium) was in-
cluded as a molecular weight standard on each gel.
A single band was visualized with UV light by
staining the gel with ethidium bromide if the PCR
was positive.
Positive controls:
A blood sample taken from
a patient with S. aureus bacteremia and another on
from a patient with E.coli bacteremia were used
as a positive control.
Negative control:
A mixture of all reagents used
for DNA extraction and DNA extracted from sterile
blood culture was processed as negative control.
DNA sequencing:
Genotypic identification of bacteria by sequence
analysis of the 16S rRNA gene was performed in
Germany. All positive PCR products were se-
quenced with the fluorescent dye terminator cycle
sequencing kit as described in the kit for BigDye
3.0 3100 (Applied Biosystems (ABI), Jena LAB,
Germany). The resulting sequence product was
purified, using ethanol/acetate, dissolved in forma-
mide, and separated on an ABI PRISM 3100 Ge-
netic Analyser (ABI). Both strands were sequenced,
stored, edited and aligned.
Consensuses between the two strands were
made in Auto Assembler computer program. The
generated text file was used for nucleotide–nucle-
otide similarity search in BLAST.
Statistical methods:
According to PCR results patients were classi-
fied into two groups: PCR positive (+ve) and PCR
negative (-ve). These 2 groups were compared by
Chi-square test or Fischer’s exact test when appro-
priate for Qualitative data and by Mann-Whitney
U test for continuous data. Sensitivity, specificity,
positive predictive value (PPV) and negative pre-
dictive value (NPV) were calculated considering
the blood culture results the gold standard. Odds
ratio(s) for association of PCR positivity with
abnormality in blood findings and clinical features
of the studied patients were calculated with 95%
confidence interval (95% CI). In all tests,
p
value
was considered significant if less than 0.05.
294
Early diagnosis of Neonatal Sepsis: The Role of 16S rRNA
Results
Fifty neonates suspected of having sepsis were
included in the study. Males represented 68% of
cases, median birth weight was 1970g (range=
940-4300g), and median gestational age was 36
weeks (range= 27-39 weeks). 66% of our neonates
were delivered vaginally and 42% had their onset
of symptoms within 72 hours of admission. The
median length of stay was 17 days (range= 3-65
days) and 42% of our cases did not survive to
discharge.
Neonates were categorized into 2 groups ac-
cording to PCR results taken upon admission.
Group 1 included neonates with positive PCR
(n=17) and group 2 included those with negative
PCR (n=33). The base line characteristics of stud-
ied neonates are shown in Table (1). There was
no significant difference between the two groups
regarding gender, birth weight, gestational age,
mode of delivery, onset of symptoms, the length
of stay, or outcome.
Table (2) shows provisional diagnosis of new-
born infants admitted to the unit. 36% of our cases
were admitted with a diagnosis of RDS, 22% with
pneumonia and 18% with suspected sepsis.
Risk factors, clinical and laboratory findings
were summarized in Tables (3,4). Hyperthermia,
feeding intolerance and abdominal distension were
significantly higher in neonates with positive PCR
(
p
=0.01, <0.01, 0.029 respectively). Other clinical
manifestations showed no significant difference
among both groups. Further analysis showed a
significant association between increasing number
of symptoms and PCR positivity (
p
=0.002). Odds
ratio for having 2 or more of significant findings
and PCR positivity was 12.1 (95% CI=3-48).
Regarding maternal or neonatal risk factors of
sepsis, there was no significant difference between
both groups.
As for hematological manifestations, WBC
count was significantly lower in patients with
positive PCR {median= 6.85/mm
3
(range= 2-
22/mm
3
) vs. 10.50/mm
3
(range= 4-27/mm
3)}.
Similarly IT ratio was higher in patients with
positive PCR {median= 0.205 (range= 0.1-0.4)
vs. 0.18 (range=0.1-0.4)}. Leucopenia and throm-
bocytopenia were also commoner among the same
group with a statistically significant difference
(
p
=0.002 and 0.04 respectively). Also we found
a significant association between increasing num-
ber of abnormal hematological findings and PCR
positivity (
p
=0.006,
p
for ordinal correlation=
0.01). Odds ratio for having 2 or more abnormal
hematological findings and PCR positivity was
3.77 (95% CI= 1.2-12.3).
Blood culture results (N=61 samples):
Blood culture was positive in 12 samples
(19.7%), Klebsiella was predominant (6 patients)
followed by coagulase-negative Staphylococci (4
patients) and Enterobacter and Diphtheroids sp,
each one sample. Two cultures yielded Candida
species and were considered as negative bacterial
cultures.
Molecular analysis: (N=61 samples) (Figs. 1,2):
Using broad-range PCR amplification samples,
18 (29.5%) samples were positive for 16S rRNA.
In one case two amplicons were detected; only
one could be sequenced, this case was denoted
'double sequence' in the result. The PCR in this
study showed sensitivity, specificity, positive
predictive value and negative predictive value of
91.7%, 85.4%. 61 % and 97.6% respectively. Ac-
curacy of this molecular test was 86.7%.
Comparison of PCR/sequencing and culture results
(Tables 5, 6, 7):
Concordant culture-positive and PCR-positive/
sequencing results was proved in 10 samples with
91 % agreement between genotypic identification
by sequencing with phenotypic identification of
bacteria grown in blood culture bottles. The percent
homology of the overlapping sequence for the
best matches were also determined.
While concordant culture-negative, PCR-
negative samples were 42 samples. This means
that 69% of neonatal blood samples lacked detect-
able levels of bacteria by both culture and PCR
analyses.
Culture-positive, PCR-negative discrepant
samples: a single blood sample positive by culture
but negative by PCR. The culture isolate was
identified as a coagulase-negative Staphylococcus
(CoNS) species.
PCR-positive, culture-negative discrepant sam-
ples: Seven specimens were PCR positive with
sterile culture. The sequencing-based identification
of these isolates included: Enterobacter cloacae,
Staphylococcus carnosus, Proteus mirabilis, Hae-
mophilus influenzae, Streptococcus pyogenes and
Klebsiella sp. One sample yielded insufficient
DNA.Clinical data of these patients are presented
in Table (7).
Ismail M. El-Hawary, et al.
295
Table (1): Base line characteristics of studied neonates in relation to PCR.
Positive
PCR (n=17)
Negative
PCR (n=33)
Total
(n=50)
p
value
Gender:
Male
14 (82.4%)
20 (60.6%)
34 (68.0%)
0.12
Female
3 (17.6%)
13 (39.4%)
16 (32.0%)
Birth weight (g) (median, range):
1500 (940-4300)
2300 (980-4200)
1970 (940-4300)
0.34
>_
2500 (N, %)
6 (35.3%)
15 (45.5%)
21 (42.0%)
0.49
<2500 (N, %)
11 (64.7%)
18 (54.5%)
29 (58.0%)
Gestational age in weeks (median, range):
33 (27-38)
36 (27-39)
36 (27-39)
0.20
>
37 (N, %)
6 (35.3%)
15 (45.5%)
21 (42.0%)
0.49
<37 (N, %)
11 (64.7%)
18 (54.5%)
29 (58.0%)
Mode of delivery:
Vaginal (N, %)
10 (58.8%)
23 (69.7%)
33 (66.0%)
0.44
C-section (N, %)
7 (41.2%)
10 (30%)
17 (34.0%)
Onset of suspected sepsis:
Within 72 hours
7 (41.2%)
14 (42.4%)
21 (42.0%)
0.93
After 72 hours
10 (58.8%)
19 (57.6%)
29 (58.0%)
*Length of stay (median, range)
27 (9-49)
17 (3-65)
17 (3-65)
0.265
Mortality
7 (41.2%)
14 (42.4%)
21 (42.0%)
0.93
Data are given as median and range or number and percentage (%) as indicated.
Table (2): Provisional diagnosis in relation to PCR.
Provisional
diagnosis
Positive
PCR (n=17)
Negative
PCR (n=33)
Total
(n=50)
p
value
RDS
8 (47.1%)
10
(30.3%)
18
(36.0%)
0.24
Pneumonia
3 (17.6%)
8
(24.2%)
11
(22.0%)
0.73
Neonatal sepsis
3 (17.6%)
6
(18.2%)
9
(18.0%)
1.0
HIE
1 (5.9%)
3
(9.1%)
4
(8.0%)
1.0
TTN
0 (.0%)
3
(9.1%)
3
(6.0%) 0.54
MAS
1 (5.9%)
0
(.0%)
1
(2.0%) 0.34
CHD
0 (.0%)
2
(6.1%)
2
(4.0%) 0.54
Hypoglycemia
0 (.0%)
1
(3.0%)
1
(2.0%)
1.0
Hyperbilirubinemia
1 (5.9%)
0
(.0%)
1
(2.0%) 0.34
Data are given as number and percentage (%).
RDS
: Repiratory distress syndrome.
HIE
:
Hypoxic ischemic encephalopathy.
TTN
: Transient tachypnea of the newborn.
MAS
:
Meconium aspiration syndrome.
CHD : Congenital heart disease.
296
Early diagnosis of Neonatal Sepsis: The Role of 16S rRNA
Table (3): Risk factors and clinical manifestations in relation to PCR.
Positive
PCR (n=17)
Negative
PCR (n=33)
Total
(n=50)
p
value
Maternal risk factors:
PROM
3 (17.6%)
3 (9.1%)
6 (12.0%)
0.40
Clinical manifestations:
General:
Hypothermia
9 (52.9%)
10 (30.3%) 19 (38.0%) 0.12
Hyperthermia
5 (29.4%)
1 (3.0%)
6 (12.0%)
0.01 (S)*
Respiratory system:
Respiratory distress
14 (82.4%)
25 (75.8%)
39 (78.0%)
0.73
Apnea
8 (47.1%)
7 (21.2%)
15 (30.0%)
0.059
Cardiovascular system:
Tachycardia
2 (11.8%)
10 (30.3%) 12 (24.0%)
0.18
Poor capillary refill
7 (41.2%)
13 (39.4%)
20 (40.0%)
0.90
Gastrointestinal system:
Abdominal distention
10 (58.8%)
9 (27.3%)
19 (38.0%)
0.029 (S)*
Feeding intolerance
12 (70.6%)
9 (27.3%)
21 (42.0%)
<0.01 (HS)**
Central nervous system:
Lethargy
8 (47.1%)
15 (45.5%)
23 (46.0%)
0.91
Irritability
5 (29.4%)
6 (18.2%)
11 (22.0%)
0.48
Seizures
4 (22.2%)
4 (9.3%)
8(16%)
1.0
Neonatal risk factors:
Mechanical ventilation (MV)
12 (70.6%) 17 (51.5%)
29 (58.0%)
0.20
Duration of MV in days (median, range)
11 (7-23)
7 (2-35)
9 (2-35)
0.168
Chest tube insertion
3 (17.6%)
3 (9.1%)
6 (12.0%)
0.40
Umbilical catheter
1 (5.9%)
4 (12.1%)
5 (10.0%)
0.65
TPN
2 (11.8%)
1 (3.0%)
3 (6.0%)
0.26
Data are given as number and percentage (%) as indicated.
(S)*
: Significant.
(HS)**
: Highly significant.
PROM
: Prolonged rupture of membranes.
MV
:
Mechanical ventilation.
TPN
: Total parenteral nutrition.
Table (4): Laboratory findings in relation to PCR.
Laboratory findings
Positive
PCR (n=17)
Negative
PCR (n=33)
Total
(n=50)
p
value
WBCs/mm
3
(median, range)
6.85 (2-22)
10.50 (4-27)
9.60 (2-27)
0.019 (S)*
IT ratio (median, range)
0.205 (0.1-0.4)
0.14 (0.1-0.4)
0.180 (0.1-0.4)
0.03 (S)*
Platelets count (median, range)
118 (5-405)
186 (24-533)
180 (5-533) 0.067
Abnormal results:
WBC >20000/mm
3
1 (5.6%)
3 (7.0%)
4 (6.6%)
1.0
WBC <5000/mm
3
7 (38.9%)
2 (4.7%)
9 (14.8%)
0.002 (HS)**
IT ratio
≥
0.2
11 (61.1%)
17 (39.5%)
28 (45.9%)
0.12
Thrombocytopenia (<150,000/mm
3
)
10 (55.6%)
12 (27.9%)
22 (36.1%)
0.04 (S)*
Data are given as median and range or number and percentage (%) as indicated.
(S)*
: Significant.
(HS)
: Highly significant.
Birth
weight
in grams
Gestational
age
in weeks
Provisional
diagnosis
Sequencing
result
Clinical
presentation
RDS
RDS
RDS
Pneumonia
Pneumonia
3200
38
Enterobacter cloacae
2300
36
Staphylococcus carnosus
1500
32
Proteus mirabilis
1500
36
Haemophilus influenzae
1300
30
Streptococcus pyogenes
1260
30
Unsuccessful
RDS
38
Klebsiella sp.
3370
Pneumonia
Respiratory distress, tachycardia,
lethargy
Respiratory distress, tachycardia,
lethargy, feeding intolerance,
abdominal distension
Hypothermia, respiratory distress,
apnea, feeding intolerance
Respiratory distress, feeding
intolerance, abdominal distension
Respiratory distress, tachycardia,
poor capillary refill, seizures,
feeding intolerance, abdominal
distension
Hypothermia, respiratory distress,
lethargy, feeding intolerance
Respiratory distress, poor capillary
refill, lethargy
Ismail M. El-Hawary, et al.
297
Table (5): Agreement of genotypic identification by sequencing with phenotypic identification of
bacteria grown in blood culture bottles.
Sequencing result
%
Homology
Culture
result
Culture x
sequencing
Klebsiella pneumoniae
98
Klebsiella sp.
Concordant
Klebsiella pneumoniae
98
Klebsiella sp
Concordant
Klebsiella sp.
98
Klebsiella sp.
Concordant
Klebsiella sp.
96
Klebsiella oxytoca
Concordant
Klebsiella pneumoniae
98
Klebsiella sp.
Concordant
Corynebacterium sp.
98
Corynebacterium sp.
Concordant
Staphylococcus warneri
98
CoNS
Concordant
Staphylococcus warneri
98
CoNS
Concordant
Enterobacter cloacae
98
Enterobacter sp.
Concordant
Staphylococcus carnosus +unsuccessful
97
CoNS
Concordant
Streptococcus sp.
96
Klebsiella sp.
Discordant
% homology
: The percent of the overlapping sequence for the best match.
CoNS
: Coagulase-negative staphylococci.
Table (6): Microorganism identification by only one method (PCR/sequencing
or culture).
PCR result
Sequencing
result
%
Homology
Culture
– –
CoNS
1
– –
Candida sp.
–
Candida sp.
+
Enterobacter cloacae
98
+
Staphylococcus carnosus
96
–
+
Proteus mirabilis
96
–
+
Haemophilus influenzae
98
–
+
Streptococcus pyogenes
100
–
+
Unsuccessful
–
+
Klebsiella sp.
98
–
1: This case was considered as culture contamination as microorganism was isolated
only once among the 3 cultures associated with negative PCR assay.
Table (7): Provisional diagnosis, clinical presentation and sequencing results in neonates with negative
blood culture.
RDS: Respiratory distress syndrome.
298
Early diagnosis of Neonatal Sepsis: The Role of 16S rRNA
Fig. (1): Results of PCR of samples 29-42. Samples number:
32, 38 and 42 were positive. In sample number 38;
two amplicons were detected;" double sequence".
Fig. (2): Results of samples 43-56. Samples number: 48, 52
and 54 were positive. Sample number 52 with cul-
ture-negative/PCR-positive results lacked sufficient
DNA.
Discussion
Despite major advances in neonatal medicine,
differentiating sepsis from other illnesses in the
neonatal period is often difficult, even more, it is
challenging. Early clinical manifestations are often
nonspecific and can be easily confused with other
non infectious causes. In our study we found a
variety of symptoms and signs in most newborn
infants suspected of having sepsis. The most com-
mon were respiratory distress, lethargy, feeding
intolerance, poor capillary refill, abdominal disten-
sion and temperature instability. When we analyzed
clinical data according to the presence of positive
PCR, hyperthermia, feeding intolerance and ab-
dominal distension were found to be significant
among neonates with proven sepsis. Several studies
have tried to identify different clinical manifesta-
tions in neonates with sepsis; however, they sug-
gested that clinical criteria alone were unable to
distinguish infected infants from uninfected ones
[13,17]
. Early diagnosis of sepsis is usually made
when there is high index of suspicion and the
presence of the above signs should alert our doctors
that these infants may be septic, and urgent labo-
ratory tests should be done.
Many studies have focused on the use of hema-
tological parameters in the diagnosis of neonatal
sepsis
[18]
. In our study the presence of 2 or more
abnormal hematological findings was significantly
higher among infants with positive PCR. Simple
tests such as total white blood cell count and IT
ratio may have a role, however, a wide variability
in the diagnostic accuracy of leukocyte indices in
neonatal sepsis exists, especially the band count
and its derived immature/total neutrophil ratio
[19]
.
The etiological diagnosis of neonatal bacterial
sepsis is based mainly on conventional blood cul-
ture, however, obtaining sufficiently large amounts
(
>_
0.5ml) of blood for culture from neonates is
difficult, and it usually takes 48-72 hours to obtain
a preliminary positive result. In addition, low-level
bacteremia (colony count 0-4CFU/ml) can not be
detected in small volume blood cultures
[20]
.
A faster and more sensitive method for the
detection and specification of bacteria or bacterial
DNA would allow earlier treatment. Moreover,
rapid bacterial identification reduces costs by
decreasing the length of hospitalization and con-
serving hospital resources
[5,20]
.
Molecular tech-
niques are particularly suitable for clinical micro-
biology because they do not require culture, they
have rapid turnaround times, and digital genetic
information can be stored in a database for epide-
miological studies. Many studies evaluated the use
of broad-range PCR for detection of 16S rRNA
for early diagnosis of neonatal sepsis
[7,8,9]
. In
most of these studies, compared to culture, PCR
demonstrated excellent analytical specificity and
negative predictive value.
In our study, the rate of culture proven sepsis
was 19.7%. With the molecular method of broad
range 16S rRNA PCR, the detection of bacteria
i
mproved to 29.5%. PCR revealed sensitivity,
specificity, positive predictive value and negative
predictive value of 91.7%, 85.4%. 61% and 97.6%
respectively, while the accuracy of this test was
86.7%. So, compared to culture, the 16S rRNA
PCR demonstrated a high negative predictive value
for ruling out neonatal sepsis. The lower specificity
of PCR in our study is because of all the 7 cases
with positive PCR but sterile cultures being true
positives and not false positives and can be ex-
plained by the possible low-level bacteremia and
humbled sensitivity of gold standard due to small
volume samples.
In clinical practice using PCR as a test with
high negative predictive value could help doctors
to stop unnecessary antibiotics as soon as PCR
results are available.
Ismail M. El-Hawary, et al.
299
Few studies used PCR product sequencing tool
to increase the diagnostic data and genotypic iden-
tification of the neonatal sepsis
[21]
. In our study,
we tried to increase the diagnostic capabilities of
our clinical microbiology laboratory in a clinical
setting, by rRNA sequencing which was not applied
before in our hospital.
Using PCR only, 11 samples were concordant
with blood culture results, but application of product
sequencing proved only 10 cases of concordance
with 90.7% agreement between two methodologies.
Other studies that used PCR alone proved higher
levels of agreement 8,9 so PCR sequencing could
be an essential step for verifying PCR results.
All cases with negative blood culture and pos-
itive PCR are very important as PCR sequencing
could detect pathogens that were not detected by
routine blood culture. In our study seven samples
showed no growth by culture but were identified
by PCR sequence analysis. Possible contamination
was possible in one isolate only that is Staphylo-
coccus carnosus which is well known to be apatho-
genic. However, the other six samples revealed
pathogenic organisms known to cause neonatal
sepsis such as Enterobacter cloacae, Proteus mira-
bilis, Haemophilus influenzae, Streptococcus pyo-
genes and Klebsiella sp 10 and these patients ended
up with the diagnosis of sepsis based on clinical
and laboratory judgments. Furthermore, PCR se-
quencing can be time saving. In our study, the
turnaround time for PCR was 6 hours and sequence
analysis was 12 hours, as a result, identification
of the organism can be achieved within 18 hours.
Identification of bacteria by PCR sequence
analysis is still costly. However, the costs of pos-
sible mortality or morbidity due to improper or
lack of identification of an isolate, incorrect diag-
nosis, or excessive use of antibiotics should also
be considered. As a start, use of PCR sequence
analysis can be limited to neonates with the most
significant risk factors and clinical manifestations
suggestive of sepsis or neonates with positive PCR.
The availability of DNA sequencers in terms of
cost, methodologies, and technology in large peri-
natal centers or university hospitals where high
load of neonates are seen should be considered.
We concluded from this work that PCR can be
used to rule out sepsis. Combining PCR and se-
quence analysis can provide additional diagnostic
data that cannot be obtained with the use of broad-
range PCR or routine laboratory tests alone. In the
setting of clinically suspected sepsis, a primary
decision could be made when the PCR results are
completed, and before the next antibiotic dose the
genotypic identification from the sequencing could
be available.
References
1-
RAHMAN S., HAMEED A., ROHGANI M.T. and ULLAH
Z.: Multidrug resistant neonatal sepsis in Peshawar,
Pakistan. Arch. Dis. Child Fetal Neonatal Ed., 87 (1):
F52-54, 2002.
2-
OSRIN D., VERGNANO S. and COSTELLO A.: Serious
bacterial infections in newborn infants in developing
countries. Curr. Opin. Infect Dis., 17: 217-224, 2004.
3-
OHLIN A., BACKMAN A., BJORKQVIST M., et al.:
Real-time PCR of the 16S-rRNA gene in the diagnosis
of neonatal bacteremia. Acta Paediatrica, 97: 1376-1380,
2008.
4-
GRIFFIN M.P. and MOORMAN J.R.: Toward the Early
Diagnosis of Neonatal Sepsis and Sepsis-Like Illness
Using Novel Heart Rate Analysis. Pediatrics, 107 (1): 97-
104, 2001.
5-
KELLOGG J.A., FERRENTINO F.L., GOODSTEIN
M.H., LISS J., SHAPIRO S.L. and BANKERT D.A.:
Frequency of low level bacteremia in infants from birth
to two months of age. Pediatr. Infect. Dis. J., 16: 381-
385, 1997.
6-
JORDAN J.A. and DURSO M.B.: Comparison of 16S
rRNA gene PCR and BACTEC 9240 for detection of
neonatal bacteremia. J. Clin. Microbiol., 38: 2574-2578,
2000.
7-
BROZANSKI B.S., JONES J.G., KROHN M.J. and JOR-
DAN J.A.: Use of polymerase chain reaction as a diag-
nostic tool for neonatal sepsis can result in a decrease in
use of antibiotics and total neonatal intensive care unit
length of stay. Perinatology, 26: 688-692, 2006.
8-
JORDAN J.A. and DUESO M.B.: Real-time polymerase
chain reaction for detecting bacterial DAN directly from
blood of neonates being evaluated for sepsis. J. Mol.
Diagn, 7: 575-581, 2005.
9-
JORDAN J.A., DURSO M.B., BUTCHKO A.R., JONES
K.G. and BROZANSKI B.S.: Evaluating the near-term
infant for early onset sepsis: progress and challenges to
consider with 16rDNA polymerase chain reaction testing.
J. Mol. Diagn, 8: 357-363, 2006.
10-
QIAN Q., TANG Y.W., KOLBERT C.P., TORGERSON
C.A., HUGHES J.G., VETTER E.A., HARMSEN W.S.,
MONTGOMERY S.O., COCKERILL F.R. III and PERS-
ING D.H.: Direct identification of bacteria from positive
blood cultures by amplification and sequencing of the
16S rRNA gene: evaluation of BACTEC 9240 instrument
true-positive and false-positive results. J. Clin. Microbiol.,
39: 3578-3582, 2001.
11-
FONTANA C., FAVARO M., PELLICCIONI M., PIS-
TOIA E. S. and Favalli C.: Use of the MicroSeq 16S rRNA
gene-based sequencing for identification of bacterial
isolates that commercial automated systems failed to
identify correctly. J. Clin. Microbiol., 43: 615-619, 2005.
12-
CLARRIDGE J.: Impact of 16S rRNA gene sequence
analysis for identification of bacteria on clinical microbi-
ology and infectious diseases. Clin. Microbiol. Rev., 17:
840-862, 2004.
300
Early diagnosis of Neonatal Sepsis: The Role of 16S rRNA
13-
FANAROFF A.A., KORONES S.B., WRIGHT L.L.,
VERTER J., POLAND R.L., BAUER C.R., TYSON J.E.,
PHILIPS J.B. III, EDWARDS W., LUCEY J.F., CATZ
C.S., SHANKARAN S. and OH W.: Incidence, presenting
features, risk factors and significance of late onset septi-
cemia in very low birth weight infants. The National
Institute of Child Health and Human Development Neo-
natal Research Network. Pediatr. Infect. Dis. J., 17: 593-
598, 1998.
14-
GONZALEZ B.E., MERCADO C.K., JOHNSON L.,
BRODSKY N.L. and BHANDARI V.: Early markers of
late-onset sepsis in premature neonates: clinical, hemato-
logical and cytokine profile. J. Perinat Med., 31: 60-68,
2003.
15-
SMULIAN J.C., BHANDARI V., CAMPBELL W.A.,
RODIS J.F. and VINTZILEOS A.M.: Value of umbilical
artery and vein levels of interleukin-6 and soluble intra-
cellular adhesion molecule-1 as predictors of neonatal
hematologic indices and suspected early sepsis. J. Matern
Fetal Med., 6: 254-259, 1997.
16-
Manual of clinical Microbiology 9
th
. Ed., editor in chief
Patrick Murray. American Society for Microbiology Press.
Washington D.C., 2007.
17-
BHANDARI V., WANG C., RINDER C. and RINDER
H.: Hematologic Profile of Sepsis in Neonates: Neutrophil
CD64 as a Diagnostic marker. Pediatrics, 121 (1): 129-
134, 2008.
18-
RODWEL R.L., LESLIE A.L. and TUDEHOPE D.I.:
Early diagnosis of neonatal sepsis using a hematologic
scoring system. J. Pediatr., 112: 761-767, 1988.
19-
DA SILVA O., OHLSSON A. and KENYON C.: Accuracy
of leukocyte indices and C-reactive protein for diagnosis
of neonatal sepsis: a critical review. Pediatr. Infect. Dis.
J., 14: 362-6, 1995.
20-
CONNEL T.G., RELE M., COWLEY D., BUTTERY J.P.
and CURTIS N.: How reliable is a negative blood culture
result? Volume of blood submitted for culture in routine
practice in a children's hospital. Pediatrics, 119: 891-896,
2007.
21-
RUPPENTHAL R.D., PEREIRAFS, CANTARELLI V.V.
and SCHRANK I.S.: Application of broad range bacterial
PCR amplification and direct sequencing on thediagnosis
of neonatal sepsis. Brazilian Journal of microbiology, 36:
29-35, 2005.
