Direct detection of Enterococcus faecium, Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa and Escherichia coli from whole blood with the use of
the T2Bacteria Panel allows early antimicrobial stewardship intervention
in patients with sepsis
Pavel Drevinek1*, Jakub Hurych1,
Milena Antuskova1, Jan Tkadlec1, Jan
Berousek2, Zuzana Prikrylova2, Jiri
Bures2, Jaromir Vajter2, Martin
Soucek3, Jan Masopust3, Vendula
Martinkova4, Jaroslava Adamkova4,
Veronika Hysperska5, Eliska Bebrova1
1 Department of Medical Microbiology,
2nd Faculty of Medicine, Charles University, Motol
University Hospital, Prague, Czech Republic;
pavel.drevinek@lfmotol.cuni.cz,
jakub.hurych@lfmotol.cuni.cz,
milena.antuskova@lfmotol.cuni.cz,
jan.tkadlec@lfmotol.cuni.cz,
eliska.bebrova@fnmotol.cz
2 Department of Anesthesiology and ICM,
2nd Faculty of Medicine, Charles University, Motol
University Hospital, Prague, Czech Republic;
jan.berousek@fnmotol.cz,
zuzana.prikrylova@fnmotol.cz,
jiri.bures@fnmotol.cz,
jaromir.vajter@fnmotol.cz
3 Department of Internal Medicine,
2nd Faculty of Medicine, Charles University in Prague,
Motol University Hospital, Prague, Czech Republic;martin.soucek@fnmotol.cz,
jan.masopust@fnmotol.cz
4 3rd Department of Surgery,
1st Faculty of Medicine, Charles University, Motol
University Hospital, Prague, Czech Republic;
vendula.martinkova@fnmotol.cz,
jaroslava.adamkova@fnmotol.cz
5 Department of Rehabilitation and Sports Medicine,
2nd Faculty of Medicine, Charles University, Motol
University Hospital, Prague, Czech Republic;
veronika.hysperska@fnmotol.cz
*Correspondence: pavel.drevinek@lfmotol.cuni.cz; Tel.: +42022443 5390
Abstract
In the microbiological diagnosis of bloodstream infections (BSI), blood
culture (BC) is considered the gold standard test despite its
limitations such as low sensitivity and slow turnaround time. A new
FDA-cleared and CE-marked platform utilizing magnetic resonance to
detect amplified DNA of the six most common and/or problematic BSI
pathogens (Enterococcus faecium , Staphylococcus aureus ,Klebsiella pneumoniae , Acinetobacter baumannii ,Pseudomonas aeruginosa and Escherichia coli ) is available
and may shorten the time to diagnosis and potentially improve
antimicrobial utilization.
Whole blood samples from hospitalized patients with clinical signs of
sepsis were analyzed using the T2Bacteria Panel (T2Biosystems) and
compared to simultaneously collected BC. Discrepant results were
evaluated based on clinical infection criteria, combining supporting
culture results and opinion of treating physicians.
A total of 55 samples from 53 patients were evaluated. Sensitivity and
specificity of T2Bacteria panel was 94% (16 out of 17 detections of
T2Bacteria-targeted organisms) and 100%, respectively, with 36.4% (8
of 22) causes of BSI detected only by this method. The T2Bacteria Panel
detected pathogens on average 55 hours faster than standard BC.
In our study, 9 of 15 patients with positive T2Bacteria Panel result
received early-targeted antibiotic therapy and/or their antimicrobial
treatment was modified based on T2Bacteria Panel findings. Given the
high reliability, faster time to detection and easy workflow, the
technique qualifies as a point of care testing approach.
Keywords: Bacteremia, Sepsis, T2MR, blood culture, rapid
diagnostics, antimicrobial stewardship
1. Introduction
Bloodstream infections (BSIs) are characterized by high morbidity and
mortality, consequences that have been linked to delays in early and
accurate administration of antimicrobials [1].
Timely detection of BSI pathogens is an unmet need of today’s medical
microbiology practice. It remains largely dependent on blood culture
(BC) [2] which performance is being continuously improved with new
protocols [3] and new approaches for pathogen identification and
determination of their antimicrobial susceptibility [4,5]. However,
BC still has its inherent limitations including the requirements for
large amounts of blood or the long turnaround time. Low sample volumes
and exposure to antimicrobials prior to BC sample collection
considerably increase the risk of false-negative BC results [6] .
Despite these limitations, BC remains the gold standard and the first
line tool for detecting BSIs.
In an effort to improve BSI management, several innovative diagnostic
techniques have been developed to optimize direct pathogen detection
from whole blood samples [6,7]. Some of them were introduced with
limited success [8] or with inconclusive results of their validation
studies [9, 10]. In this study we report our real-world experience
with the T2Bacteria Panel (T2Biosystems, USA) which utilizes T2 magnetic
resonance (T2MR) to detect six bacterial BSI pathogens
(Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa andEscherichia coli , commonly referred to as ESKAPEc bacteria)
directly from whole blood [11]. This culture-independent method can
greatly shorten the time to positivity and like other existing rapid
diagnostic methods, when integrated with an antibiotic stewardship
program, may reduce the time to appropriate therapy and possibly improve
infection-related outcomes such as duration of hospitalization or
mortality.
2. Materials and Methods
Study population . This prospective study was conducted between
November 2018 and April 2019 at Motol University Hospital, a tertiary
care teaching hospital with over 2200 beds (18% of them being intensive
care) and roughly 80,000 hospitalizations per year. Patients were
eligible for study enrollment if they a) were being managed by
Anesthesiology and Intensive Care Medicine Department or by intensive
care units of Internal Medicine (including Hematology) or Surgery
Departments, and b) presented clinical signs of BSI, evaluated at the
discretion of the managing physician. Initially, only the patients whose
samples were taken within standard opening hours of microbiology
laboratory (weekdays from 6 am to 4 pm) were included to ensure
immediate processing of blood samples. In order to intensify patient
recruitment, eligibility hours in April 2019 were expanded from 6 am to
midnight (except weekends).
Laboratory methods . All blood samples were subjected to both
culture and the T2Bacteria Panel testing. BCs were processed using
BACTEC FX™ Automated Blood Culture System (Becton Dickinson, USA).
Positive flagged BCs were primed for Gram stain. Subsequent pathogen
identification from positive BC bottles was accomplished with
matrix-assisted laser desorption/ionization time-of flight mass
spectrometry (Bruker Daltonics, USA) with the first attempt for
identification made after 2 hours of pre-incubation on solid media
[12].
Direct detection of six bacterial pathogens on the T2Dx instrument
(T2Biosystems, USA) requires 4 mL whole blood specimens. During
processing on the T2Dx, bacteria are concentrated directly in whole
blood, then lysed to release the DNA. After amplification, target
amplicon is hybridized with superparamagnetic particles and then
detected by T2MR [13]. The results are available in as few as 3.5
hours. According to the manufacturer’s instructions for use the
T2Bacteria Panel is indicated as an aid in the diagnosis of bacteremia
and results should be used in conjunction with other clinical and
laboratory data.
All results were called to the managing clinician by the medical
microbiologist. Results were reported as target detected or target not
detected for each of the 6 bacterial targets and accompanied with the
optional antimicrobial stewardship in case of positivity.
Study Endpoints and Statistical Analysis . The primary outcomes
of this study were sensitivity and specificity of the T2Bacteria Panel,
which were calculated using positive BC and/or T2Bacteria Panel results
for a T2Bacteria-targeted organism as the reference. Species detected by
BC but not included in the T2Bacteria Panel were excluded from
sensitivity and specificity calculation. Similar to Nguyen et al.and De Angelis et al. , we developed infection criteria in order
to resolve any discrepancies between T2Bacteria Panel and BC [11,
14]. “Proven BSI” was defined as a positive BC (excluding potential
BC contaminants) using a concurrently drawn specimen. T2Bacteria Panel
results were determined to be “Probable BSIs” when the
T2Bacteria-detected organism was isolated within 21 days from another BC
specimen collected at a different time or from a clinical sample taken
from another site (such as the abdominal fluid, urine, or
bronchoalveolar lavage), indicating a plausible cause of infection.
Finally, “Possible BSI” was defined as a negative BC, but a positive
T2Bacteria Panel result in the absence of supporting culture data if the
T2Bacteria Panel detected organism was a plausible cause of disease.
Proven, probable and possible BSIs were included in final sensitivity
and specificity calculation.
We also quantified the time to species identification between the BC and
T2Bacteria Panel and noted any antibiotic stewardship interventions made
as a result of the T2Bacteria Panel result. The time to species
identification was defined as the hour interval between the time when a
BC or T2Bacteria Panel sample was received in lab and the time when the
ID results were reported. Continuous variables were presented as means
and standard deviations, and were compared using a 2 sample t-test, as
appropriate. Categorical variables were presented as counts and
percentages, and were compared using an adjusted Wald confidence
interval. Differences were considered to be statistically significant
for p <0.05. Statistical analyses were performed by using
Minitab®19 Statistical Software (Minitab, LLC.).
3. Results
During the study period, 55 samples were obtained and tested from 53
unique patient cases (Figure 1). Testing of blood samples by using
T2Bacteria Panel resulted in the recovery of 16 positive detections from
15 samples yielding a 27% rate of positivity (15 of 55). All 16
detections were considered proven or probable cause of BSI based on the
pre-specified definitions for BSI (see Methods). In total, 23 culture
findings were recovered from 20 BC samples. As 9 of them were deemed
potential normal skin flora contaminants and not treated, only 14
findings from 14 BC samples were considered to be clinically relevant.
However, 5 of them were not identifiable using the T2Bacteria Panel
(Figures 1 and 2) and thus excluded from sensitivity and specificity
calculation. Since probable BSIs were assumed to be true positives that
were missed by concurrent BC, sensitivity and positive predictive value
was calculated to be 94% (95% CI, 71.1%-99.9%) and 100%
respectively (95% CI, 82.9%-100%). Only one unexplained
false-negative T2Bacteria detection (E. coli ) was identified in
one sample that was T2Bacteria Panel-positive for another pathogen
(S. aureus ). There were 40 concordant negative detections
(in regard to T2Bacteria Panel targeted organisms), yielding a
specificity of 100% (95% CI, 92.4%-100%) and a negative predictive
value of 98% (95% CI, 86.3%-99.9%).