A Study using ETGA® Technology, Cognitor® Minus, to Predict the Negative
Results of Blood Cultures after 12 hours Incubation in the Paediatric Setting.
Joanne Bullivant, Sheffield Teaching Hospitals NHS Trust.
5
17:00:00
Time results available comparison
A 9am sample cut off in the laboratory would provide
a result to the ward by 12.30pm. If using a 9am cut off
23 of 124 samples would have a result later than the 36
hour blood culture. Times ranged from 13 minutes slower to 3 hours and 20 minutes slower. A 12pm sample
cut off for testing would provide a result to the ward by
3.30pm. Using a 12pm cut off 10 of 124 samples results
would have been slower by the Cognitor® assay compared with 36 hour blood culture result. Times ranged
from 17 minutes slower to 3 hours and 25 minutes slower.
Conclusions
• The Cognitor® assay provides reliable negative
blood culture results within 15 hours after receipt of
the sample within the laboratory, NPV 100%.
• The assay only takes 3 hours to perform with approximately 2 hours hands on time so it can realistically fit into the laboratory workflow if clinicians
think it would be a useful adjuvant.
• The results of the Cognitor® assay do have the potential to reduce bed nights and antimicrobial use
by providing a result on average 10 hours earlier
than the gold standard blood culture.
• The clinical implications of obtaining a more rapid
result will help to screen out ‘well’ babies allowing;
prevention of administration of unnecessary antibiotics and earlier discharge whilst allowing medical
staff to focus attentions on those that are ill.
0
112
Cognitor® NOT
Negative result
11*
1
12
Total
123
1
124
PCR
Template
16:00:00
Figure 5: Time taken for sample to be received in the
laboratory.
112**
Substrate
Modification
Mean time taken for a
sample to be received in
the lab after inoculation:
5 hours 39 minutes and
16 seconds, range 27
minutes to 15 hours 18
minutes. Median time
taken: 4 hours 52 minutes
and 30 seconds. Only 40
out of 124 samples tested
were received in the lab
within 4 hours of being
taken. Main delay due to
transport, neonatal wards
are 4 miles away, off site.
Time Difference from Sample taken to Reciept
in the Laboratory
Total
Cognitor®
Negative
15:00:00
0
14:00:00
Figure 2: A schematic overview of the Cognitor® assay.
10
13:00:00
Microbial
Lysis
15
Blood Culture
Positive at
36 hours
Bibliography
Sample
Clean-up
20
Blood Culture
Negative at
36 hours
Table 1: Summary of results
Specimen
25
12:00:00
•
Figure 4: Reasons for Blood Culture Request.
Group B Streptococcus was documented in 18 out of
124 samples tested.
11:00:00
•
Results
Suspected Sepsis
10:00:00
•
To determine whether Cognitor® Minus can
produce reliable negative results from blood
cultures within 15 hours of specimen receipt in
the laboratory.
To evaluate how Cognitor® would fit into the
laboratory workflow.
To establish whether it has the potential to
reduce bed nights and antimicrobial use.
To determine the clinical implications of obtaining
a more rapid negative result.
Method
After a minimum of 12 hours incubation 1ml of specimen was
removed from each blood culture bottle, promptly returning the
bottles to the blood culture system for continued testing, each
bottle was removed from the analyser for less than 10 minutes,
ensuring no automated readings were missed. The specimen
was added to a 1.5ml Eppendorf, from reagent pack 1 and the
remaining drop of sample in the syringe was inoculated onto
a chocolate agar plate and incubated for 48 hours at 37ºC in
aerobic conditions, to check for any microbial growth. Ideally
samples would be tested after exactly 12 hours incubation,
during this study samples were batched and results analysed
as if tested at 12 hours or appropriate to the laboratory working
day. To ensure maximum use from each kit in the study the
samples were tested in batches of 6, there is no upper limit
regarding length of incubation only a minimum of 12 hours is
required. If the test was to be introduced into the laboratory the
aim would be to test samples as close to 12 hours incubation
as possible to ensure maximum effect from the results. It is
unrealistic to test each sample at exactly 12 hours due to time
of receipt of the samples by the laboratory which can occur at
any time of the day.
Manufacturer’s methodology and guidelines followed as per kit
instructions for Cognitor® Minus, User Manual Version 4 (2).
Maternal Pyrexia
09:00:00
•
Premature Rupture of Membranes
Antibiotics started
Maternal Sepsis IV Antibiotics
08:00:00
Aims
Pre Term
25
07:00:00
•
•
Respiratory Distress
Maternal Group B Streptococcus
06:00:00
•
Jaundice
63
05:00:00
•
Meconium Admission
3
15
Sample Size
In total 124 samples paediatric blood culture samples were
tested, sample size calculation based on using 0.05 for alpha,
0.8 and power, with 1 (100%) sensitivity in group 1 (blood
culture results) and 0.95 (95%) sensitivity in group 2 (Cognitor®
result), provided a guide of 122 samples that would need to be
tested by both methods (the paired comparison). As there is no
comparison study currently published the sample size is used
as a guide and will be reviewed based on results generated.
Figure 3: The Cognitor Kit
2 3
04:00:00
•
Suspected maternal sepsis requiring intravenous
antibiotics 24 hours before to 24 hours after
delivery
Invasive group B streptococcus infection in a
previous infant
Maternal colonisation with group B streptococcus
(in high vaginal swab or urine)
Rupture of membranes >24 hours before birth if
term and 18 hours if preterm
Spontaneous preterm labour (<37 weeks)
Chorioamnionitis
(defined
as
maternal
temperature >37.5ºC on two occasions more
than one hour apart, or >38ºC on one occasion,
maternal white blood cell count >20 or foul
smelling liquor
1
11
03:00:00
•
1
02:00:00
Risk factors for EOS
Patient Inclusion
Blood culture bottles are received throughout the day
from neonatology and are loaded onto an automated
blood culture analyser system. Automated analyser
used was the BD BACTEC™ FX Blood Culture System
and BD BACTEC™ PEDS PLUS™/F Medium bottles,
blood is inoculated into bottles on the wards and sent
to the laboratory for analysis. Blood cultures from
post-natal neonatology wards were incubated as per
hospital guidelines for five days on the BD BACTEC™ FX
Automated Blood Culture System. Samples were targeted
at neonates with possible risk factors to develop EOS.
01:00:00
Figure 1: Bactec FX Instrument and Blood Culture
Bottle
Materials and methods
00:00:00
Cognitor® Minus is a kit that uses enzymatic template
generation and amplification (ETGA®) technology
to detect DNA polymerase in replicating organisms
in blood culture samples. The assay is designed to
screen out negative samples and provide results
within 15 hours of receipt of the sample at the
laboratory. After a minimum of 12 hours incubation
on an automated blood culture analyser, 1ml of
specimen is removed from the blood culture bottle
for testing.
ETGA® technology detects the activity of nucleic acid
modifying enzymes, Cognitor® Minus exploits this to
detect microorganisms by measuring microbial DNA
polymerase activity. Using this method Cognitor®
Minus should be able to detect any microorganism
because DNA polymerase activity is common to all
living things. The assay does not identify organisms
but will tell you if a sample is likely to contain any.
At Sheffield Teaching Hospital NHS Foundation Trust
(STH) Medical Microbiology and Paediatric medical
staff have identified a possible use for the Cognitor®
Assay.
Neonates with risk factors for early onset sepsis (EOS)
require a 36 hour negative blood culture result before
antibiotic therapy can be stopped and the patient
can be discharged. Cognitor® could reduce the wait
for this time by up to 21 hours allowing clinicians to
make earlier decisions about patient management
preventing the use of unnecessary antibiotics and
freeing up bed space on already overstretched
maternity services.
Frequency
Introduction
*To aid analysis of data and comparison of the 2 tests
Cognitor® results that were not negative have been
analysed all together. A result other than negative would
not be acted on and the 36 hour blood culture result
required. Not negative results include, not determined
and unresolved results (where the internal control failed
to amplify) 2 of 11 results were unresolved and 9 of 11
results were not determined.
**Includes 13 negative results where the internal control
did not amplify in the negative control. Upon analysis
of the data it was decided that these results would be
accepted as negative as the internal control amplified in
other samples and the positive control was positive on
each run, demonstrating amplification. The results would
be used in conjunction with other laboratory results on the
ward to form a clinical decision.
Cognitor® User Manual. Instructions for Use. Momentum Bioscience. http://www.momentumbio.co.uk Becton
Dickinson www.bd.com/uk National Institute for Health and Care Excellence (NICE). Neonatal Infection:
antibiotics for prevention and treatment. Clinical guideline. Published: 22 August 2012 nice.org.uk/guidance/
cg149 Public Health England. UK Standards for Microbiology Investigations. Detection of Carriage of Group
B Streptococci. Bacteriology. B58. Issue No. 3. Issue Date 19.06.2015.
B 37. Investigation of Blood Cultures (for organisms other than Mycobacterium species). UK Standards for
Microbiology Investigations. Issue 8. 04.11.2014. Public Health England.