Ashok K. Yadav, C.G. Wilson, P.L. Prasad and P.K.
Menon
From the Department of Pediatrics, Armed Forces
Medical College, Pune , Maharashtra-411 040, India.
Correspondence to Dr. (Surgeon Lieut. Commander)
Ashok K. Yadav, Pediatrician, Indian Naval Hospital Ship Jeevanti,
Vasco Da Gama, Goa-403 802, India.
E-mail:
[email protected]
Manuscript received: September 8, 2003, Initial
review completed: December 2, 2003;
Revision accepted: February 2, 2005.
Clinical diagnosis of sepsis in new born
infants is not easy because symptoms and signs are non-specific.
There is no laboratory test with 100% specificity and sensitivity,
search has continued for a reliable test. Blood culture has been
the gold standard for confirmation of diagnosis but the results of
the test are available only after 48-72 hours. The neonates with
"risk factors" for neonatal sepsis are thus treated with
broad-spectrum antibiotics and require prolonged hospitalization.
The alterations of laboratory tests are
corroborative parameters to diagnose neonatal sepsis. The
sensitivity of each laboratory test is far from 100%(1-5). The
measurements of IL-1 receptor antagonist, IL-6, IL-8, are still
confined to the research laboratories(6,7). The advent of
polymerase chain reaction has made it possible to have a 100%
specific and sensitive method for diagnosis of bacterial sepsis in
a short time(8,9).
DNA sequence present in all bacteria such as
portions of the DNA encoding the 16-S- ribosomal-RNA has been used
to define an organism as a bacterium (9). Those sequences are
amplified with PCR, using an automated method allowing a rapid
diagnosis. By using Multiplex-PCR simultaneous amplification of
more than one genetic locus using more than one primer pair can be
used to differentiate various etiological agents responsible for
sepsis based on the molecular weight of the fragments
amplified(10). In this study the aim was to evaluate the molecular
diagnosis of sepsis using PCR amplification of 16 S rRNA in
newborns with risk factors for sepsis or those who have clinical
evidence of sepsis. We also compared the results of PCR with blood
culture and other markers of sepsis screen [total leucocyte count
(TLC), absolute neutrophil count (ANC), immature/total neutrophil
count ratio (I/T ratio), peripheral blood smear, micro-ESR and
C-reactive protein (CRP)].
Subjects and Methods
At level II neonatology unit of a tertiary care
referral hospital 100 newborns with 1 major or 2 minor risk
factors for sepsis as per ACOG guidelines(11) were included in the
study. The major risk factors for inclusion were: (i)
premature rupture of membrane (PROM)>24 hrs; (ii) premature
onset of labor <37 week; (iii) Chorioamnionitis; (iv)
Intrapartum maternal fever >38ºC and the minor risk factors for
inclusion were: (i) PROM >12 hrs, but <24 hrs; (ii)
prematurity; (iii) very low birth weight (<1500 gms); (iv)
intrapartum maternal fever >37.5ºC but <38ºC; (v) multiple
gestations; and (vi) apgar score at one minute <5 .
Soon after birth, 1 mL of venous blood was
drawn for blood culture and PCR each. Also 5 mL of venous blood
was collected for TLC, DLC, peripheral blood smear, micro-ESR and
CRP. The sepsis screen consisting of TLC <5 × 109/L, ANC <1.5 ×
109/L, I/T ratio >0.2, micro-ESR and CRP was carried out. The
sepsis screen was considered positive if I/T ratio >0.2 and CRP
was positive(12). The newborns developing symptoms and signs of
sepsis later (within 07 days) were also investigated by PCR and
blood culture as per the above protocol. The gold standard for
diagnosis of sepsis was positive blood culture.
The control group consisted of 30 cases of
normal neonates admitted to the postnatal ward who had neither
overt evidence of sepsis nor any of the risk factors. They were
evaluated for sepsis with sepsis screen and subjected to PCR
amplification for 16-S RNA (861 base pair).
Presence of bacterial DNA in blood samples was
evaluated by amplifying the DNA region encoding 16 rRNA (861 base
pair) using the following primers: F-5AGAGTTTGAT-CCTGGCTCAG-3’
(15) and R-5’GGACTACCAGGGTACTT AAT-3’(15). A small volume of the
sample (2µ) was mixed in the PCR reaction(12), (briefly, 50
mM KCI, 10 mM Tris-HCL (pH-8), 1.5 mM MgCl2, 200 µL each d NTP, 60
pM each primer and 2.5U Taq polymerase, in a final volume of 100
µL. The PCR was carried out using whole blood (2µL)
as the sample for DNA templates(13).
The Taq polymerase was then added and the
eppendorf tubes were placed in the Automatic Thermal Cycler. The
amplification used an initial modifying denaturation step (03 min
at 94ºC and 03 min at 55ºC for 3 times) followed by 30 cycles (1
min at 95ºC, 1 min at 54ºC and 1 min at 72º C). A final extension
cycle of 72ºC for 07min was carried out. Each PCR run was carried
out including a known positive control; the kit control DNA
produced a 600 bp band. The amplified DNA was separated by agarose
gel electrophoresis stained with ethidium bromide and visualized
under UV trans-illuminator. Analysis was carried out keeping blood
culture as the gold standard.
Results
The study population comprised of 100 newborns
who fulfilled the ACOG criteria for probable sepsis from a
population of 1923 live births at a large tertiary care hospital
who presented consecutively. The prevalence of maternal risk
factors was PROM (18%), maternal fever (10%), foul discharge per
vaginum (6%) and multiple gestations (4%).
The mean birth weight of the neonates in the
study group was 1829 ± 608 grams. The average age of diagnosis of
sepsis (based on blood culture results) was 3.6 days and ranged
from 2-7 days. In only one neonate intravascular catheter was used
and 11 (11%) newborns required some resuscitation. The mean birth
weight of the neonates in the control group was 2310 ± 516 grams.
The prevalence of neonatal risk factors was prematurity (15%),
VLBW (07%) and low APGAR score (11%).
The 06 paramecters of sepsis screen were
studied in the study as well as the control group. Of the 100
neonates in the study group I/T ratio of >0.2 and positive CRP was
observed in 24 cases (24%), 12 cases (12%) had TLC <5000/cumm,
presence of toxic granules in 6% cases and two cases each (4%)
fulfilled the criteria for ANC and micro-ESR.
Blood culture yielded positive results in 9
(9%) of the study group and had a sensitivity of 69.2% for
diagnosis of neonatal sepsis. Lumbar puncture was performed in all
septic neonates with positive blood culture and only 02 (2/9) had
abnormal cytology. All CSF cultures were sterile. In comparison
with that the sepsis screen had sensitivity of 100%, specificity
of 83.5% and negative predictive value of 100% (Table I).
PCR amplification for 861 bp was positive in 13 cases (13%). This
included 04 cases with positive sepsis screen but negative blood
culture. PCR was positive in all blood culture positive cases.
Thus PCR is proved to have sensitivity of 100%, specificity of
95.6% and negative predictive value of 100%. The lower specificity
of PCR is because of all the 04 cases with positive PCR but
sterile cultures being true positives and not false positives and
can be explained by the lower sensitivity of the gold standard. Of
the 15 neonates with positive sepsis screen but negative blood
culture, PCR was positive in 4 cases (Table II).
TABLE I
Utility of sepsis screen for diagnosis of Neonatal Sepsis.
|
Sepsis screen |
Blood culture |
| |
Positive |
Negative
|
Positive
|
9
|
15
|
Negative
|
0
|
76
|
TABLE II
Utility of PCR in Diagnosis of Neonatal Sepsis.
|
PCR |
Blood culture
|
|
Positive |
Negative |
Positive
|
9
|
04
|
Negative
|
0
|
87
|
In the study group with risk factors for
sepsis, 24 cases (24%) required antibiotics for 14-21 days and in
remaining 76 cases (76%) antibiotics were stopped based on
clinical condition, sterile blood culture and negative PCR for 861
bp bands. All the neonates had a normal outcome.
Discussion
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(14). For obvious reasons there is a tendency to over
investigate and over treat neonates for suspected sepsis. Sepsis
screen and the blood culture have been the diagnostic pillars but
their sensitivity and specificity is far from 100%(8,10,13).
In this study the incidence of early onset
neonatal sepsis is 3.1 per 1000 live births that is low compared
to the incidence reported by Mathur et al and Mondal, et al.(13-17).
Sepsis screen is a useful tool for evaluation of sepsis with the
sensitivity of 100%, specificity of 83.5% and negative predictive
value of 97.4%. Mathers, et al. and Pourcyrous, et al.
have reported similar results(17,18). With the sensitivity of
69.2% and specificity of 100%; blood culture alone would have
missed another 04 (30%) cases. The low sensitivity in this study
in comparison to 85% by Visser, et al. is likely to be
related to the use of anti-biotics in large number of mothers
(68%) in the antepartum period(19).
PCR with sensitivity of 100% and specificity of
95.6% is a useful tool because of the short time required to reach
confirmation. The PCR has shown 100% correlation with
microbiological methods in all infected cases and was positive in
four additional cases with positive sepsis screen but with sterile
blood cultures. In a similar study Laforgia, et al. studied
33 newborn infants at risk for early onset neonatal sepsis and
found 29 negative blood cultures and 4 positive blood cultures.
All cases with positive blood culture yielded 861 base pair bands.
Among cases with negative blood culture 2 cases resulted in
amplification of 861 base pair bands(13).
Unnecessary treatment with antibiotics could be
avoided in 76 cases of newborns with sterile blood culture and
negative PCR.
Contributors: CGW was involved in designing
the study, collection of data and preparation of the manuscript.
AKY was involved with the design, collection of the data, analysis
of the results and writing of the manuscript. He will act as the
guarantor of the study. PKM helped with the conduct of the PCR
analysis. PLP helped in analysis of results and drafting of the
manuscript.
Funding: None.
Competing Interests: None stated.
|
Key Messages |
|
• Sepsis screen is a useful test in clinical side room
setting for ruling out neonatal sepsis.
• Blood culture remains the gold
standard, but has limitation of delay period of 48-72 hours.
• Polymerase chain reaction with 100% sensitivity and
95.6% specificity, once available in most tertiary centers
can help in early and accurate diagnosis.
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