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%).