Abstract
The LAMP-based eazyplex® BloodScreen GN was evaluated for the detection of frequent Gram-negatives directly from positive blood culture (BC) bottles. A total of 449 BCs were analyzed. Sensitivities and specificities were 100% and 100% for Escherichia coli, 95.7% and 100% for Klebsiella pneumoniae, 100% and 100% for blaCTX-M, 100% and 100% for Klebsiella oxytoca, 100% and 99% for Proteus mirabilis, and 100% and 99.8% for Pseudomonas aeruginosa, respectively. The time to result ranged from 8 to 16 min, plus about 6 min for sample preparation. The eazyplex® BloodScreen GN is a reliable molecular assay for rapid BC testing.
Avoid common mistakes on your manuscript.
Sepsis is one of the leading causes of death in the inpatient setting, and appropriate antimicrobial therapy needs to be started in a timely manner [1, 2]. For empiric treatment, the Surviving Sepsis Campaign guideline recommends the administration of intravenous broad-spectrum antibiotics within 1 h following the diagnosis of sepsis or septic shock [3]. Recommendations also include the early targeting of antibiotics when microbiological diagnostic results are available in order to improve the clinical outcome and reduce the selective pressure for resistances [3]. Identification of bloodstream infections (BSI) still relies on blood cultures (BCs). Conventional diagnosis of positive BCs by subcultures and phenotypic antimicrobial susceptibility testing (AST) takes time and results in delays of more than 1 or 2 days before species identification and AST results are reported. MALDI-TOF mass spectrometry (MS) and molecular diagnostic assays can be used to identify the bacterial species and resistance markers directly from a positive BC bottle and are important add-on methods that allow for a more rapid optimization of antibiotic therapy [4,5,6]. Infectious Disease Society of America (IDSA) guidelines therefore recommend the use of rapid diagnostic tests (RDTs) and their integration in antibiotic stewardship programs [7]. Molecular RDTs based on fully automated random-access multiplex PCRs are very expensive. Loop-mediated isothermal amplification (LAMP) technologies offer the potential to implement fast, simple, and cost-effective molecular tools into diagnostic workflows [8, 9]. Here we evaluated the performance of the CE-labeled LAMP assay eazyplex® BloodScreen GN (Amplex Diagnostics, Gars-Bahnhof, Germany) for identification of frequent Gram-negatives from positive BCs. The assay is based on a previous in-house assay now including additional species-specific primers instead of the ubiquitous target for Enterobacterales [9]. A test strip contains ready to use lyophilized master mixes with primers for Escherichia coli, Klebsiella pneumoniae, blaCTX-M-1 group, blaCTX-M-9 group, Klebsiella oxytoca, Proteus mirabilis, and Pseudomonas aeruginosa, and an inhibition control.
Clinical samples were BCs submitted as part of routine patient care from the Jena University Hospital, the SHK Weimar, and the Waldkliniken Eisenberg, to the clinical microbiology laboratory at the Jena University Hospital between September 2019 and May 2020. Blood samples collected in BD BACTEC Plus aerobic/F and lytic/10 anaerobic/F bottles (BD Diagnostics, Heidelberg, Germany) were incubated on a BACTEC FX instrument (BD Diagnostics). An aliquot of positive BCs that revealed Gram-negative rods after Gram-staining was examined by eazyplex® BloodScreen GN and conventional diagnostics in parallel. Only the first positive BC bottle per patient, regardless of whether aerobic or anaerobic, was tested.
For LAMP testing, 5 µl of BC broth was mixed with 500 µl of resuspension and lysis fluid (RALF) and boiled for 2 min. After centrifugation at 4000 rpm for 1 min, 25 µl of the supernatant was added to each tube of the eazyplex® BloodScreen GN test strip. Tests were run on a Genie HT machine (Amplex Diagnostics) at 65 °C for 20 min. Amplification was measured by real-time fluorescence detection using a DNA intercalating dye. Data interpretation was automatically performed by the integrated eazyReport™ software (Amplex Diagnostics). Results are reported as positive in real time if the fluorescence level and the peak of the first derivative of the fluorescence curve rise above the defined thresholds.
For conventional diagnostics, BC aliquots were streaked onto Columbia sheep blood agar, chocolate agar, Schaedler agar (BD), and Drigalski lactose agar (Oxoid, Thermo Fisher Scientific, Wesel, Germany) for overnight incubation at 37 °C. Colonies were identified by Vitek MS (bioMérieux, Nürtingen, Germany). AST was performed by the determination of minimal inhibitory concentrations (MIC) using Vitek 2 (bioMérieux). Breakpoints were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. The production of extended-spectrum beta-lactamases (ESBLs) was verified by the chromogenic β LACTA™ test (Bio-Rad, Feldkirchen, Germany).
A total of 449 positive BCs representing all isolates during the study period were prospectively analyzed. The mean time to positivity of BC bottles, defined as the time between the start of incubation and the positive signal, was 12 (SD, 12.5) h. For the eazyplex® assay, preparation of one sample or two samples in parallel took 5.75 (SD, 0.75; n = 6) and 7 (SD, 0.75; n = 6) min, respectively. The time to result, defined by the threshold time of fluorescence intensity, ranged from about 8 min for P. mirabilis to 16 min for K. pneumoniae (Table 1). For 20 samples, the inhibition control of the assay was invalid. They were therefore excluded from the evaluation (4.5% of all BCs). The eazyplex® BloodScreen GN demonstrated high sensitivity and specificity for all targets (Table 2). All E. coli and K. pneumoniae cases with a positive CTX-M result were phenotypically confirmed as ESBL-producing isolates by AST (Table 3). ESBL-producing but CTX-M-negative isolates were not detected. There were two false-negative and four false-positive results in regard to the species-specific targets. The false positives were observed for P. mirabilis caused by cross-reactions with E. coli. Two false-negative results were attributed to K. pneumoniae and K. variicola as identified by Vitek MS. It should be noted that the assay cannot differentiate between both Klebsiella species (information by the manufacturer). From 79 BCs with a valid inhibition control, no species-specific test results were obtained (18.4%). Subculture identification revealed as most frequent pathogens Enterobacter cloacae complex (n = 18), Bacteroides fragilis (n = 12), Serratia marcescens (n = 6), non-typhoidal Salmonella enterica (n = 6), Citrobacter freundii complex (n = 5), and Acinetobacter baumannii complex (n = 3). Mixed infections with Gram-negatives or Gram-positives were detected in 23 BCs. In all cases, the species included in the eazyplex® assay were correctly identified. In 7 out of 10 cases of a mixed Gram-negative infection, both species were covered by the assay.
At the Jena University Hospital, piperacillin-tazobactam is primarily recommended for empiric antibiotic treatment of sepsis in patients with no documentation of prior infection or colonization with MRSA or multidrug-resistant Gram-negatives. Although the majority of ESBL-producing E. coli isolates were sensitive against piperacillin-tazobactam, escalation to meropenem based on a positive eazyplex® CTX-M result would be appropriate (Table 3). Several studies have shown that piperacillin-tazobactam appears to be inferior to carbapenems for treatment of serious infections and bacteremia caused by ESBL-producing organisms, underlining the potential benefit of RDTs to identify ESBL resistance 1 day earlier than conventional phenotypic AST [10,11,12].
De-escalation based on eazyplex® results without knowledge of phenotypic AST is unsafe. According to the ABS recommendations at our hospital, identification of CTX-M-negative E. coli and P. mirabilis would result in continuation of piperacillin-tazobactam, whereas non-ESBL K. pneumoniae and K. oxytoca are preferentially treated with a third-generation cephalosporin (Table 3) [4]. Identification of P. aeruginosa would require optimization of piperacillin-tazobactam dosing but would not allow early targeting of antibiotics unless diagnostic results and resistance information from other relevant samples are available (Table 3). When the eazyplex® assay shows no species-specific result, the presence of a SPICE organism (Serratia spp., Pseudomonas spp., Indole-positive Proteus group, Citrobacter spp., and Enterobacter spp.) or Acinetobacter spp. can be expected in most of the cases and escalation of the therapy to meropenem may be considered at least until the species is identified [13, 14].
Clinical studies on the impact of rapid BC testing on patient outcome could demonstrate that the mortality risk and length of stay decreased when a molecular RDT is implemented in ABS [15,16,17]. The results of this study show that the eazyplex® BloodScreen GN is a viable rapid assay with little workload. A further advantage of the assay is the relatively low cost for consumables (about 35 EUR per test) compared to automated random-access PCR assays that can be more than twice as expensive. MALDI-TOF MS analysis directly performed on BC aliquots has low consumable costs but is more labor-intensive due to the need for centrifugation steps and additional testing for beta-lactamases [18]. Additional costs of rapid BC testing must be balanced against the expected clinical benefit [19, 20]. RDT results more likely lead to escalation, whereas de-escalation will be rather considered when final AST results are available. In this context, it must be considered that the eazyplex® assay covers resistances against third-generation cephalosporins only by detection of blaCTX-M. Unfortunately, for this assay, no appropriate LAMP primer target that covers all species of the E. cloacae complex could be found (information by the manufacturer). Upregulated AmpC beta-lactamases in these species are responsible for inactivating third-generation cephalosporins [21, 22]. The lack of these targets may be also problematic in mixed infections [23]. Of note is the availability of eazyplex® carbapenemase test kits that can be performed as additional tools depending on the local prevalence of carbapenemase-producing Gram-negatives or the colonization status of the patient [24].
The eazyplex® BloodScreen GN has now been implemented in routine diagnostics in our laboratory. Test results are immediately reported to the clinician and/or infectiologist. Whether the ongoing antibiotic therapy is continued or escalated based on the eazyplex® result depends on additional diagnostics reports available for the patient. A treatment algorithm, which takes both the eazyplex® result and the hospital-specific antibiogram into account, has been developed (Table 4). In the context of this study, the proposed algorithm would have resulted in a recommendation of meropenem in 30% of all cases. In 22% of all cases, the use of meropenem would have been appropriate because of the isolation of Acinetobacter spp. (n = 6), ESBL-producers (n = 51), and SPICE organisms excluding P. aeruginosa (n = 38) from BCs.
In conclusion, the eazyplex® BloodScreen GN is a simple diagnostic tool for rapid BC testing. Because of the high accuracy in the identification of species and CTX-M genes, results can be used for a timely escalation of empiric therapy when indicated.
Availability of data
The dataset analyzed in this study is available from the corresponding author on reasonable request.
Code availability
Not applicable
References
Septimus EJ (2020) Sepsis perspective 2020. J Infect Dis 222(Suppl 2):S71–S73. https://doi.org/10.1093/infdis/jiaa220
Schneider JG, Wood JB, Schmitt BH, Emery CL, Davis TE, Smith NW, Blevins S, Hiles J, Desai A, Wrin J, Bocian B, Manaloor JJ (2019) Susceptibility Provision Enhances Effective De-escalation (SPEED): utilizing rapid phenotypic susceptibility testing in Gram-negative bloodstream infections and its potential clinical impact. J Antimicrob Chemother 74(Suppl 1):i16–i23. https://doi.org/10.1093/jac/dky531
Rhodes A, Evans LE, Alhazzani W, Levy MM, Antonelli M, Ferrer R, Kumar A, Sevransky JE, Sprung CL, Nunnally ME, Rochwerg B, Rubenfeld GD, Angus DC, Annane D, Beale RJ, Bellinghan GJ, Bernard GR, Chiche JD, Coopersmith C, De Backer DP, French CJ, Fujishima S, Gerlach H, Hidalgo JL, Hollenberg SM, Jones AE, Karnad DR, Kleinpell RM, Koh Y, Lisboa TC, Machado FR, Marini JJ, Marshall JC, Mazuski JE, McIntyre LA, McLean AS, Mehta S, Moreno RP, Myburgh J, Navalesi P, Nishida O, Osborn TM, Perner A, Plunkett CM, Ranieri M, Schorr CA, Seckel MA, Seymour CW, Shieh L, Shukri KA, Simpson SQ, Singer M, Thompson BT, Townsend SR, Van der Poll T, Vincent JL, Wiersinga WJ, Zimmerman JL (2016) Dellinger RP (2017) Surviving Sepsis Campaign: international guidelines for management of sepsis and septic shock. Intensive Care Med 43:304–377. https://doi.org/10.1007/s00063-017-0298-5
Claeys KC, Schlaffer KE, Heil EL, Leekha S, Johnson JK (2018) Validation of an antimicrobial stewardship-driven Verigene blood-culture Gram-negative treatment algorithm to improve appropriateness of antibiotics. Open Forum Infect Dis 5:ofy233. https://doi.org/10.1093/ofid/ofy233
Rivard KR, Athans V, Lam SW, Gordon SM, Procop GW, Richter SS, Neuner E (2017) Impact of antimicrobial stewardship and rapid microarray testing on patients with Gram-negative bacteremia. Eur J Clin Microbiol Infect Dis 36:1879–1887. https://doi.org/10.1007/s10096-017-3008-6
Zadka H, Raykhshtat E, Uralev B, Bishouty N, Weiss-Meilik A, Adler A (2019) The implementation of rapid microbial identification via MALDI-TOF reduces mortality in gram-negative but not gram-positive bacteremia. Eur J Clin Microbiol Infect Dis 38:2053–2059. https://doi.org/10.1007/s10096-019-03640-w
Barlam TF, Cosgrove SE, Abbo LM, MacDougall C, Schuetz AN, Septimus EJ, Srinivasan A, Dellit TH, Falck-Ytter YT, Fishman NO, Hamilton CW, Jenkins TC, Lipsett PA, Malani PN, May LS, Moran GJ, Neuhauser MM, Newland JG, Ohl CA, Samore MH, Seo SK, Trivedi KK (2016) Implementing an antibiotic stewardship program: guidelines by the Infectious Disease Society of America guidelines and the Society for Healthcare Epidemiology of America. Clin Infect Dis 62:e51-77. https://doi.org/10.1093/cid/ciw118
Leikeim RSM, Kesselmeier M, Löffler B, Rödel J, Höring S (2020) Diagnostic accuracy and clinical impact of loop-mediated isothermal amplification for rapid detection of Staphylococcus aureus bacteremia: a retrospective observational study. Eur J Clin Microbiol Infect Dis 39:679–688. https://doi.org/10.1007/s10096-019-03773-y
Rödel J, Bohnert JA, Stoll S, Wassill L, Edel B, Karrasch M, Löffler B, Pfister W (2017) Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures. Eur J Clin Microbiol Infect Dis 36:1033–1040. https://doi.org/10.1007/s10096-016-2888-1
Harris PNA, Tambyah PA, Lye DC, Mo Y, Lee TH, Yilmaz M, Alenazi TH, Arabi Y, Falcone M, Bassetti M, Righi E, Rogers BA, Kanj S, Bhally H, Iredell J, Mendelson M, Boyles TH, Looke D, Miyakis S, Walls G, Al Khamis M, Zikri A, Crowe A, Ingram P, Daneman N, Griffin P, Athan E, Lorenc P, Baker P, Roberts L, Beatson SA, Peleg AY, Harris-Brown T, Paterson DL, MERINO Trial Investigators and the Australasian Society for Infectious Disease Clinical Research Network (ASID-CRN) (2018) Effect of piperacillin-tazobactam vs meropenem on 30-day mortality for patients with E. coli or Klebsiella pneumoniae bloodstream infection and ceftriaxone resistance: a randomized clinical trial. JAMA 320:984–994. https://doi.org/10.1001/jama.2018.1216
Tamma PD, Han JH, Rock C, Harris AD, Lautenbach E, Hsu AJ, Avdic E, Cosgrove SE, Antibacterial Resistance Leadership Group (2015) Carbapenem therapy is associated with improved survival compared with piperacillin-tazobactam for patients with extended-spectrum β-lactamase bacteremia. Clin Infect Dis 60:1319–1325. https://doi.org/10.1093/cid/civ003
Strich JR, Heil EL, Masur H (2020) Considerations for empiric antimicrobial therapy in sepsis and septic shock in an era of antimicrobial resistance. J Infect Dis 222(Suppl 2):S119–S131. https://doi.org/10.1093/infdis/jiaa221
Moy S, Sharma R (2017) Treatment outcomes in infections caused by “SPICE” (Serratia, Pseudomonas, Indole-positive Proteus, Citrobacter, and Enterobacter) organisms: carbapenem versus noncarbapenem regimens. Clin Ther 39:170–176. https://doi.org/10.1016/j.clinthera.2016.11.025
Herrmann L, Kimmig A, Rödel J, Hagel S, Rose N, Pletz MW, Bahrs C (2021) Early treatment outcomes for bloodstream infections caused by potential AmpC beta-lactamase-producing Enterobacterales with focus on piperacillin/tazobactam: a retrospective cohort study. Antibiotics (Basel) 10:665. https://doi.org/10.3390/antibiotics10060665
Claeys K, Heil EL, Hitchcok S, Johnson JK, Leekha S (2020) Management of gram-negative bloodstream infections in the era of rapid diagnostic testing: impact with and without antibiotic stewardship. Open Forum Infect Dis 7:ofaa427. https://doi.org/10.1093/ofid/ofaa427
Walker T, Dumadag S, Lee CJ, Lee SH, Bender JM, Cupo Abbott J, She RC (2016) Clinical impact of laboratory implementation of Verigene BC-GN microarray-based assay for detection of Gram-negative bacteria in positive blood cultures. J Clin Microbiol 54:1789–1795. https://doi.org/10.1128/JCM.00376-16
Cortazzo V, D’Inzeo T, Giordano L, Menchinelli G, Liotti FM, Fiori B, De Maio F, Luzzaro F, Sanguinetti M, Posteraro B, Spanu T (2021) Comparing BioFire FilmArray BCID2 and BCID panels for direct detection of bacterial pathogens and antimicrobial resistance genes from positive blood cultures. J Clin Microbiol 59:e03163-e3220. https://doi.org/10.1128/JCM.03163-20
Torres I, Pinto C, Oltra R, Pascual T, Carbonell N, Colomina J, Tormo M, Albert E, Aguilar G, Solano C, Navarro D (2020) Early adjustment of empirical antibiotic therapy of bloodstream infections on the basis of direct identification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Gram staining results. J Infect Chemother 26:963–969. https://doi.org/10.1016/j.jiac.2020.04.019
Arroyo MA, Denys GA (2017) Parallel evaluation of the MALDI Sepsityper and Verigene BC-GN assays for rapid identification of Gram-negative bacilli from positive blood cultures. J Clin Microbiol 55:2708–2718. https://doi.org/10.1128/JCM.00692-17
Bookstaver PB, Nimmich EB, Smith TJ 3rd, Justo JA, Kohn J, Hammer KL, Troficanto C, Albrecht HA, Al-Hasan MN (2017) Cumulative effect of an antimicrobial stewardship and rapid diagnostic testing bundle on early streamlining of antimicrobial therapy in Gram-negative bloodstream infections. Antimicrob Agents Chemother 61:e00189-e217. https://doi.org/10.1128/AAC.00189-17
Cheng L, Nelson BC, Mehta M, Seval N, Park S, Giddins MJ, Shi Q, Whittier S, Gomez-Simmonds A, Uhlemann AC (2017) Piperacillin-tazobactam versus other antibacterial agents for treatment of bloodstream infections due to AmpC β-lactamase-producing Enterobacteriaceae. Antimicrob Agents Chemother 61:e00276-e371. https://doi.org/10.1128/AAC.00276-17
Tan ST, Ng TM, Chew KL, Yong J, Wu JE, Yap MY, Heng ST, Ng WHW, Wan S, Cheok SJH, Tambyah PA, Lye DC (2020) Outcomes of treating AmpC-producing Enterobacterales bacteremia with carbapenems vs. non-carbapenems. Int J Antimicrob Agents 55:105860. https://doi.org/10.1016/j.ijantimicag.2019.105860
Claeys KC, Heil EL, Pogue JM, Lephart PR, Johnson JK (2018) The Verigene dilemma: gram-negative polymicrobial bloodstream infections and clinical decision making. Diagn Microbiol Infect Dis 91:144–146. https://doi.org/10.1016/j.diagmicrobio.2018.01.012
Fiori B, D’Inzeo T, Posteraro B, Menchinelli G, Liotti FM, De Angelis G, De Maio F, Fantoni M, Murri R, Scoppettuolo G, Ventura G, Tumbarello M, Pennestrì F, Taccari F, Sanguinetti M, Spanu T (2019) Direct use of eazyplex® SuperBug CRE assay from positive blood cultures in conjunction with inpatient infectious disease consulting for timely appropriate antimicrobial therapy in Escherichia coli and Klebsiella pneumoniae blood stream infections. Infect Drug Resist 12:1055–1062. https://doi.org/10.2147/IDR.S206323
Funding
Open Access funding enabled and organized by Projekt DEAL. This work was supported by a grant from the German Federal Ministry of Education and Research (BMBF, 13N13890).
Author information
Authors and Affiliations
Contributions
K. B.: validation, formal analysis, investigation, writing—review and editing. B. E.: investigation, writing—review and editing. S. H.: conceptualization, formal analysis, writing—review and editing. L. B.: investigation, writing—review and editing. S. G.: investigation, writing—review and editing. B. L.: conceptualization, resources. C. B.: validation, writing—review and editing. J. R.: conceptualization, methodology, validation, formal analysis, investigation, resources, writing—original draft and preparation, project administration.
Corresponding author
Ethics declarations
Ethics approval
The study protocol for the evaluation of eazyplex® assays for clinical BC samples was reviewed and approved by the ethics committee of the Jena University Hospital (2019-1352-Material).
Consent to participate
The ethics committee did not require a written consent from patients.
Consent for publication
All the authors have given their consent for publication of the study.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Bach, K., Edel, B., Höring, S. et al. Performance of the eazyplex® BloodScreen GN as a simple and rapid molecular test for identification of Gram-negative bacteria from positive blood cultures. Eur J Clin Microbiol Infect Dis 41, 489–494 (2022). https://doi.org/10.1007/s10096-021-04383-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10096-021-04383-3