The clinical impact of implementing GenMark ePlex blood culture panels for around-the-clock blood culture identification; a prospective observational study

Abstract

Background: Implementing rapid molecular blood culture diagnostics in the clinical management of sepsis is essential for early pathogen identification and resistance gene testing. The GenMark ePlex blood culture panels offer a broad microbial spectrum with minimal hands-on time and approximately 1.5 h to result. Therefore, ePlex can be utilized at times when the clinical microbiology laboratory is unavailable.

Methods: From 23 October 2019 to 30 December 2019, consecutive non-duplicate positive blood cultures signalling microbial growth at the 24 h/7 days-a-week available clinical chemistry laboratory between 9 pm and 7 am were analysed with ePlex. All blood cultures were transported to the microbiology laboratory the following day for conventional identification and antibiotic susceptibility testing.

Results: We used ePlex to test 91 blood cultures, of which 86 had confirmed microbial growth. Eighty-one were positive for ePlex target pathogens. The ePlex results were in complete agreement with conventional methods in 72/81 (88.9%) of cases and available within a median of 10.9 h earlier. Resistance gene targets (11 mecA and 1 CTX-M) were concordant with phenotypic susceptibility in all cases. In 18/86 (20.9%) of the patient cases, there was an opportunity to optimize antimicrobial therapy based on the ePlex result. The ePlex result affected clinical decision-making in 4/86 (4.7%) of the cases and reduced the average time to effective antimicrobial therapy by 8.9 h.

Conclusions: Our implementation of ePlex is a feasible option to attain around-the-clock blood culture identification in many hospitals. It can significantly reduce time-to-pathogen identification and have an impact on clinical decision-making.

Keywords:

• Bloodstream infection
• GenMark ePlex
• Molecular identification
• Molecular rapid diagnostic testing

Introduction

Early administration of antibiotic therapy is critical in sepsis management [1]. Sepsis is one of the leading causes of death and morbidity and a significant challenge in most hospitals owing to extended hospitalisation and associated high costs [2–4]. Furthermore, antibiotic resistance is drastically rising, which increases the risk of ineffective empirical antimicrobial therapy [5]. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used for blood culture identification, and the technique can also be applied directly to blood culture pellets without prior subculture to further reduce the time to result. However, direct MALDI-TOF MS requires access to a microbiology laboratory and trained personnel and is also limited by an overall sensitivity of approximately 80% [6,7].

Molecular rapid diagnostic testing (mRDT) allows for rapid, easy-to-use identification of bacteria in blood cultures [8,9]. In recent years, several systems have become commercially available [10]. The latest system, GenMark ePlex blood culture panels, combines multiplex PCR with electrochemical detection technology [11]. Two panels are available, BCID-GP (20 Gram positive targets and four resistance genes) and a BCID-GN (22 Gram negative targets and six resistance genes). A fungal panel (BCID-FP) is also available, but both bacterial panels include a pan-Candida target. ePlex has a turnaround time of 1.5 h and has been reported as highly concordant with reference diagnostics, between 89 and 96% [12–14].

Blood culture identification is only available during the daytime in many microbiological laboratories. At other times, the blood cultures remain unattended until the next day, resulting in a potential delay of 15 h. The use of a satellite incubator may mitigate the delay, and the blood cultures are then transferred to the microbiology laboratory the following day for conventional diagnostics.

GenMark ePlex blood culture panels offer a broad spectrum of pathogens and resistance genes and can be performed by non-specialized personnel, making it suitable for implementation at times when the microbiology laboratory is unavailable. There is currently a lack of prospective studies evaluating the impact of ePlex testing on time-to-pathogen identification and the subsequent effect on clinical decision-making. We hypothesized that by implementing ePlex at times when the microbiology laboratory is unavailable, we could achieve close to a 24 h/7 days-a-week performance of blood culture diagnostics, resulting in a significant clinical impact for patients.

Materials and methods

The study was performed at Västerås Central Hospital, a 404-bed tertiary hospital in the county of Västmanland, Sweden, serving a population of 270,000 inhabitants. In this hospital, blood cultures (BacT/ALERT FA Plus Aerobic and FN Plus Anaerobic; bioMérieux, France) obtained after 4 pm are incubated in a satellite incubator (BacT/ALERT VIRTUO Culture System, bioMérieux, France) at the clinical chemistry laboratory, which is available 24 h/7 days-a-week. Positive blood cultures are then transferred to the clinical microbiology laboratory the following day for conventional blood culture diagnostics between 8 am and 5 pm.

From 23 October 2019 to 30 December 2019, consecutive non-duplicate positive blood cultures identified at the clinical chemistry laboratory between 9 pm and 7 am were prospectively analysed with ePlex as part of a new clinical routine (Figure 1).

Figure 1. Workflow of blood culture diagnostics.

The choice of the panel (BCID-GP or BCID-GN) was based on Methylene blue stain microscopy identification of cocci or rods . Methylene blue stain was chosen over Gram stain as it is easier to learn and faster to perform. Detection of a pan Gram positive or pan Gram negative target resulted in sequential testing of both panels. The fungal panel was not used. The regular laboratory biomedical analysts conducted all analyses as per the manufacturer’s instructions and within 1 h of indication of microbial growth. All preparations were conducted in a Class II biosafety cabinet. A time limit for the aerobic blood culture positivity was set to 72 h to avoid the risk of Brucella sp. or Francisella tularensis in the sample. No additional personnel were employed during the study period. At 7 am, all positive blood cultures were transported to the microbiology laboratory for culture-based identification, MALDI-TOF MS, and antibiotic susceptibility testing (AST).

All positive blood cultures were sub-cultured on agar plates (nonselective blood agar, Columbia blood agar base (Oxoid, UK) containing 5% horse blood, Chocolate agar, GC-agar base (Becton Dickinson, USA) with Iso Vitalex and gentian violet blood agar Columbia blood agar base (Oxoid, UK) containing 5% sheep blood and Methylrosaniline solution 15 mg/L. The blood agar plates were incubated in a 35 °C aerobic atmosphere. Chocolate agar and Gentian violet blood agar plates were incubated in a 35 °C atmosphere with 5% CO₂. Additionally, FN Plus anaerobic cultures were spread on fastidious anaerobic agar (Neogen, USA) with 5% horse blood and incubated in an anaerobic atmosphere at 35° C. Single spotted colonies were identified with MALDI-TOF MS (VITEK MS bioMérieux, France). From 13 November 2019, rapid AST was also conducted on all blood cultures by direct inoculation on Mueller–Hinton Agar 2 (Becton Dickinson, USA) using 100–150 µL of blood. AST was conducted at 4, 6, and 8 h, with breakpoints adapted to each incubation time as per EUCAST [15].

Direct-MALDI-TOF MS was performed on a single blood culture in each set, and FA Plus was prioritized if both cultures were positive. An in-house method was used. First, an aliquot of 200 µL of 5% saponin solution was added to 1 ml of blood and centrifuged at 13,000 rpm for 1 min. Then, the supernatant was discarded, and the pellet was washed with 1 ml of purified water. Following re-centrifugation, the supernatant was discarded, and the pellet was dried for 2 min before being distributed to four spots of the MALDI-TOF MS (VITEK MS bioMérieux, France).

The ePlex result, except for the findings of Staphylococcus epidermidis and Staphylococcus sp., was communicated to the attending physician by a printed copy that was faxed to the emergency department, followed by a confirmation phone call. Due to technical reasons, the ePlex result could not be reported in the medical records. The hospital´s high proportion of coagulase-negative staphylococci (CoNS) contaminants, approximately 20% of positive blood cultures, and low level of clinically relevant CoNS infections motivated the decision not to include CoNS in the reporting routine for ePlex results.

The ePlex result was reported as preliminary, awaiting confirmation by standard blood culture diagnostics. The attending physician was responsible for performing the appropriate antimicrobial adjustments. As per routine, an infectious disease specialist was always available by telephone for consultation and antibiotic stewardship.

The time difference between the ePlex result and the first available result to the species level of culture-based identification or direct MALDI-TOF were recorded. The target resistance genes detected by ePlex were categorized as true or false positive based on the results of disc diffusion tests on the agar plates and as false-negative if resistance testing by conventional methods could detect target resistance.

The impact of the ePlex result on clinical decision-making was evaluated by a review of the patients’ medical records. Positive blood cultures were categorized as true bloodstream infections (BSIs) or contamination based on number of positive cultures and type of organism [16]. Modifications to the antimicrobial therapy were specified as being the initiation of antimicrobial treatment or the de-escalation or broadening of the empirical treatment

An infectious disease specialist defined the optimal antimicrobial therapy for each patient case. The impact on clinical decision-making was defined as the difference in the time between the initiation of ePlex-guided optimal antimicrobial therapy and the first result obtained from conventional diagnostics that would have enabled the same clinical decision, either by pathogen identification or AST.

The study was approved by the Swedish Ethical Review Board (DNR 2019-01261).

Results

We used ePlex to test 91 consecutive non-duplicate blood cultures (signalling microbial growth) from 89 unique patients (median age 75 years, interquartile range [IQR] 64.5–82.0 years), (Figure 2). Of them, five blood cultures that were negative by ePlex were subsequently negative by standard Gram stain microscopy and culture. Non-target pathogens were cultured in five blood cultures (one each of Achromobacter sp., unidentified anaerobic Gram negative, unidentified Gram positive coccus, Actinotignum schaalii, and non-coli Escherichia sp.) In 8 patients there were additional pathogens identified by standard culture in blood cultures obtained within 24 h of the blood cultures included in study (five BSIs and two contaminants).

Figure 2. Flowchart of ePlex results.

There were 81 blood cultures positive for ePlex target pathogens (Table 1), and they were deemed by a medical microbiologist to be concordant with the standard culture method in 72/81 (88.9%) cases, being 92.0% and 89.7% concordant for single Gram positive and single Gram negative bacteria, respectively. The nine non-concordant results included two by ePlex invalid results (Escherichia coli and Salmonella sp.), five where no targets were detected by ePlex (one each of E. coli, Staphyloccocus haemolyticus, Candida albicans, Cutibacterium acnes and Lactobacillus sp.), one failure to use the appropriate ePlex panel (E. coli, pan Gram negative target positive, but Gram positive panel used twice) and a polymicrobial blood culture result (Streptococcus mitis and two S. epidermidis) where ePlex correctly identified the S. mitis but failed to identify S. epidermidis.

Table 1. ePlex target pathogen detection compared with standard culture.

Parameter	Standard culture identification results (n)	ePlex identification results (n)	%Concordance
Total species identification	81	72	89%
Single Gram positive	50	46	92%
Staphylococci
Staphylococcus aureus	13	13
Staphylococcus epidermidis	14	14
Coagulase-negative staphylococci	8	7^a
Streptococci
Streptococcus pneumoniae	2	2
Streptococcus pyogenes	2	2
Streptococcus agalactiae	2	2
Streptococcus sp.	5	4^b
Enterococci
Enterococcus faecium	1	1
Other
Lactobacillus sp.	1	0
Corynebacterium	1	1
Cutibacterium acnes	1	0
Single Gram negative	29	26	90%
Enterobacteriaceae
Escherichia coli	14	12
Klebsiella oxytoca	2	2
Klebsiella pneumoniae	2	2
Enterobacter cloacae complex	1	1
Enterobacter sp.	2	2
Citrobacter sp.	1	1
Proteus mirabilis	2	2
Serratia marcescens	1	1
Salmonella sp.	1	0
Pseudomonas aeruginosa	1	1
Other
Bacteroides fragilis	2	2
Candida sp.	1	0
Polymicrobial	1 (3 sp.)	0 (1/3 sp.)^c
Resistance gene target
mecA (S. epidermidis 8, CoNS 1, S. aureus 2)	11	11
CTX-M (E. coli 1)	1	1

^a Failed to identify Staphylococcus haemolyticus.

^b Failed to identify Streptococcus bovis.

^c Identified S. mitis but failed to identify two S. epidermidis.

No false-positive ePlex results were detected, yielding a specificity of 100%. The resistance gene targets, 11 mecA (eight S. epidermidis, one CoNS 1 and two Staphylococcus aureus) and one CTX-M positive E. coli, were concordant with phenotypic susceptibility in all cases, and there were no cases where the target resistance genes were unidentified by ePlex.

Using Methylene blue stain microscopy, the biomedical analysts were able to determine the correct ePlex panel to use first in 82/86 (95.3%) of the cases. Contaminants were identified in 21/86 (24.4%) of the blood culture results.

The positive ePlex results were available a median of 10.9 h (IQR 8–14.5) before identification to the species level by standard culture/MALDI-TOF. As the findings of CoNS were not sent to the emergency department, 52 ePlex results were made available to the attending physicians.

At the time of the ePlex result, 8/52 (15.4%) patients were not receiving any antibiotics or were receiving empirical treatment with little to no effect on the identified pathogen. Due to the ePlex result, appropriate antibiotic therapy was initiated in 2 of these patients (vancomycin for methicillin-resistant Staphylococcus aureus [MRSA] and benzylpenicillin for Streptococcus agalactiae) and two others received broadened antibiotic therapy (cefotaxime to meropenem for CTX-M-positive Escherichia coli and oral pivmecillinam to cefotaxime for E. coli). In these cases, the ePlex impact on clinical decision-making reduced the time to appropriate therapy by an average of 8.9 h compared with when conventional diagnostics (microscopy, culture/MALDI-TOF or AST) could have yielded the same clinical decision. In the remaining four cases, the ePlex result did not impact the clinical decision-making. For two of these cases, there was no comment in the medical records, and it was unclear whether the attending physician ever received the result. In the other two cases, the result was commented on in the medical records, however, there was no change to the ineffective antibiotic therapy (cefotaxime for Pseudomonas aeruginosa and piperacillin/tazobactam for Enterococcus faecium).

ePlex identified two cases of Streptococcus pyogenes, and while both were receiving effective antibiotics (cefotaxime and cloxacillin), improvements to the therapy were possible by the addition of clindamycin, and intravenous immunoglobulin could have been administered in one of the cases due to septic shock, but was not.

De-escalation of the broad empiric antibiotic therapy would have been possible in 8/52 (15.4%) of the patient cases (five cases of methicillin-susceptible S. aureus, two cases of S. mitis and one case of S. agalactiae) but this was not performed until the result of the standard method was available.

To summarize, ePlex had an impact on clinical decision-making in 4/86 (4.7%) of the patient cases. In total, the results had the potential to impact the treatment in 18/86 (20.9%) of the cases based on the optimal antimicrobial therapy decided for each patient case.

Discussion

This study presents an impact on clinical decision-making in 4.7% of patient cases and an average reduction of time to optimal antimicrobial therapy by 8.9 h, by implementing ePlex at times when the microbiological laboratory was unavailable. Given that the delay of effective antibiotic administration in septic shock is associated with an substantial decrease in survival for each hour [17], our results imply that implementing ePlex may reduce morbidity and mortality in septic patients. The limitations of this study include the small sample size and the lack of a control group, which may have exaggerated the impact on clinical decision-making, as other factors also influence the decision to initiate or modify treatment, such as clinical deterioration.

In our study, 15.4% of the patient cases, where the ePlex result was made available to the attending physician, were receiving inadequate antibiotic therapy, which correlates with the lower range of previous studies [18–20]. ePlex only impacted the clinical decision-making in 50% of these cases, leaving room for further improvement. As the clinicians become more accustomed to receiving the ePlex results, the responsiveness may increase over time, and the addition of user-defined comments to help guide antibiotic therapy could also increase the impact. Although importing the ePlex results into the laboratory module of the patients’ medical records is preferred, it was not possible during the study period due to technical reasons.

There was an opportunity for either initiation, optimisation or de-escalation of antimicrobial treatment in 20.9% of the patient cases, based on ePlex results, but this was performed in only 4.7% of the cases. Possible explanations for the lack of de-escalation include the result being reported as preliminary and that not all blood cultures from each patient were tested. Previous studies have also reported difficulties in promoting antibiotic de-escalation during night-time, as the attending physicians were hesitant to contact the antibiotic stewardship team regarding nonurgent inquiries [21,22]. Mandatory antibiotic stewardship consultation for all ePlex results would likely promote de-escalation but would also increase costs. Efforts to promote early de-escalation in clinically stable patients may increase the impact of the ePlex result. The low prevalence of polymicrobial blood cultures, which was 1.2% of the cultures tested with ePlex and 9.3% of the patient cases if all blood cultures obtained in proximity were included, reflect the mixed population at the study hospital. In a hospital with predominantly surgical patients, the incidence has been reported to be more than 10% [23]. ePlex´s failure to identify S.epidermidis in the polymicrobial infection may be due to the shorter incubation time at the time of analysis compared to when the blood culture was subjected to standard diagnostics.

The high prevalence of CoNS contaminants increased the cost of ePlex implementation, and as the results were not communicated to the attending physicians, the findings did not benefit the patients. The purpose of withholding the CoNS-results was to avoid confusion and unwarranted actions as the results were conveyed to the attending physician as something that needed urgent attention.

Due to low levels of antibiotic resistance, septic patients in Sweden generally receive empirical antibiotic treatment that do not cover MRSA, vancomycin-resistant enterococci, or Enterobacteriaceae expressing extended-spectrum beta-lactamases. Resistance gene detection was the basis for the broadening of empirical treatment in two of the patient cases. The recently published Merino trial [24] reported the superiority of meropenem over piperacillin/tazobactam in treating extended-spectrum beta-lactamase-producing Enterobacteriaceae, and the inclusion of CTX-M in the BCID-GN panel enables an early switch to carbapenem-based treatment while maintaining carbapenem-sparing strategies in empirical treatment. In Sweden, the proportion of MRSA in blood cultures positive for S. aureus is 1.8% [25], and MRSA coverage is consequently often abstained in empirical therapy. The ePlex result of MRSA in one patient, unknown to be an MRSA carrier, enabled the switch to vancomycin 12 h before conventional diagnostics were able to diagnose MRSA. In other settings, where empirical coverage of MRSA is routinely administered, the result may instead assist in the earlier de-escalation of antibiotic therapy.

ePlex targets comprised 94.2% of the cultured pathogens, and non-target pathogens were mainly regarded as contaminants. Gene resistance detection had both 100% sensitivity and specificity in our study. The ePlex was, therefore, a good choice of mRDT system for the diversity of pathogens and resistance genes in our setting.

To date, commercially available mRDT-systems include the BioFire FilmArray blood culture identification panel (FA-BCID) and the Verigene Gram positive and Gram negative blood culture panels (BC-GP and BC-GN, respectively). Both systems have reported reliability in the identification of pathogens and resistance genes comparable with the ePlex system [9,26,27]. BioFire FA-BCID is a single panel that incorporates both Gram negative and Gram positive targets, which enables testing without prior microscopy. Although FA-BCID includes only 19 bacterial pathogens, 74/86 (86.0%) of the positive blood cultures in this study had pathogens included among the targets. The panel lacks Gram negative resistance targets other than KPC, but an updated panel, including more pathogens and resistance genes, is under development. In our study, Methylene blue-stain microscopy was not an obstacle when implementing ePlex, and the advantage of a single panel may not compensate for the loss of targets. Verigene BC-GP and BC-GN also depend on microscopy and target fewer pathogens and resistance genes, therefore, offer few advantages.

Key factors to consider when implementing mRDT include the biomedical analysts’ skill level and work shifts. At the study hospital, only six biomedical analysts cover the 9 pm–7 am work shift at the clinical chemistry laboratory, two at a time. Training in Methylene blue-stain microscopy and the ePlex system was, therefore, easy to accomplish for the biomedical analysts in this shift. Without prior experience in Methylene blue-stain microscopy, the biomedical analysts identified the correct panel in almost all cases.

mRDT is a significant advancement in blood culture diagnostics and a critical component in high-quality sepsis management. The Infectious Diseases Society of America has recommended the prompt implementation of mRDT in routine care [28], but differences in access to the microbiology laboratory, laboratory workflow, and patient populations affect the optimal implementation strategy. Future advancements in MALDI-TOF-based antimicrobial resistance testing may further limit the use of mRDT to times when MALDI-TOF is unavailable.

The need to combine mRDT with antimicrobial stewardship programmes to direct the appropriate therapy has been frequently reported [29,30] and is confirmed by our study. Our findings demonstrated that ePlex substantially impacted clinical decision-making in several patient cases. We believe that our approach is a feasible option to attain around-the-clock blood culture diagnostics in many hospitals. Future studies, with larger patient samples and in settings with a higher prevalence of resistant microorganisms and including control groups are necessary to confirm the generalizability of our results.

Acknowledgments

We thank the bio-medical analysts; Helena Wästling, Roseangela des Olivera Santos, Katrina Överkvist, Bodil Gustavsson, Catarina Sillén and Maria Birro Pettersson for performing the ePlex analyses in this study.

Disclosure statement

No potential conflict of interest was reported by the author(s).