scholarly journals Evaluation of CARBA PAcE, a novel rapid test for detection of carbapenemase-producing Enterobacterales

2020 ◽  
Author(s):  
Janko Sattler ◽  
Anne Brunke ◽  
Axel Hamprecht
Keyword(s):  
False Negative ◽  
Rapid Test ◽  
Content Type ◽  
Columbia Blood Agar ◽  
Fast Detection ◽  
The Poor ◽  
Colorimetric Test ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
Moderate Sensitivity

Introduction. Carbapenemase-producing Enterobacterales (CPE) are an increasing threat to global health. Fast detection is crucial for patient management and outbreak control. Hypothesis/Gap statement. Recently, a new commercial colorimetric test, CARBA PAcE, was released that has not yet been scientifically evaluated. Aim. Our goals were to evaluate the performance of CARBA PAcE using a large variety of different CPE. Methodology. CARBA PAcE was challenged with 107 molecularly characterized CPE and 53 non-CPE controls. Isolates were grown on Mueller-Hinton agar (MHA); in the case of a false-negative result, isolates were additionally inoculated on Columbia blood agar (CBA) and CARBA PAcE was repeated. The test was performed according to the manufacturer’s protocol. Results. CARBA PAcE showed an overall sensitivity and specificity of 72 % [confidence interval (CI) 62–80 %] and 91 % (CI 79–97 %), respectively, when isolates were grown on MHA. With growth on CBA, detection improved (especially of metallo-β-lactamases), resulting in an extrapolated sensitivity of 89 % (CI 81–94 %) for all carbapenemases and 96 % (CI 89–99 %) for the four major carbapenemases (NDM, OXA-48-like, KPC, VIM). Conclusion. CARBA PAcE is a simple and very rapid test for the detection of CPE which performs well for the major carbapenemases when isolates are grown on CBA. Laboratories should be aware of the limitations of this assay, such as moderate sensitivity when isolates are grown on more challenging agars such as MHA and the poor detection of some rare carbapenemases (e.g. IMI, OXA-58).

scholarly journals Multiplex Immunochromatographic Detection of OXA-48, KPC, and NDM Carbapenemases: Impact of Inoculum, Antibiotics, and Agar

2018 ◽  
Vol 56 (5) ◽  
Author(s):  
Ahmad Saleh ◽  
Stephan Göttig ◽  
Axel G. Hamprecht
Keyword(s):  
False Negative ◽  
Rapid Test ◽  
Zinc Content ◽  
Inhibition Zone ◽  
Content Type ◽  
Negative Results ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
Lateral Flow Tests ◽  
The Impact

ABSTRACT For the rapid detection of carbapenemase-producing Enterobacteriaceae (CPE), immunochromatographic lateral flow tests (ICT) have recently been developed. The aim of this study was to assess the new multiplex ICT Resist-3 O.K.N. and to investigate if it can be performed directly from susceptibility testing plates. Additionally, the impact of the inoculum and carbapenem disks on sensitivity and specificity was evaluated. The new ICT was challenged using 63 carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 51 carbapenemase producers. It was assessed under five different conditions directly from Mueller-Hinton agar (MHA): 1 μl or 10 μl of inoculum harvested in the absence of antibiotic pressure or 1 μl taken from the inhibition zone of either an ertapenem, imipenem, or meropenem disk. The sensitivity of the ICT was 100% for OXA-48-like and KPC carbapenemases and 94.4% for the NDM carbapenemase with the 1-μl inoculum. When harvested adjacent to a carbapenem disk, the sensitivity increased to 100%. Additionally, with zinc-supplemented MHA, both the sensitivity increased and the NDM band became visible faster (mean time, 8 ± 3.9 min for MHA compared to 1.9 ± 1.5 min for MHA plus zinc; P = 0.0016). The specificity of the ICT was 100%. The Resist-3 O.K.N. ICT is a sensitive and rapid test for the detection of three highly prevalent carbapenemases. However, false-negative results for NDM can occur. We recommend an inoculum of 1 μl that is harvested adjacent to an ertapenem or meropenem disk and the use of agars with sufficient zinc content to achieve the best performance.

scholarly journals Correlation between Mutations inliaFSRof Enterococcus faecium and MIC of Daptomycin: Revisiting Daptomycin Breakpoints

2012 ◽  
Vol 56 (8) ◽  
pp. 4354-4359 ◽  
Author(s):  
Jose M. Munita ◽  
Diana Panesso ◽  
Lorena Diaz ◽  
Truc T. Tran ◽  
Jinnethe Reyes ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Cell Envelope ◽  
Strong Association ◽  
Brain Heart Infusion ◽  
Regulatory System ◽  
Content Type ◽  
Initial Event ◽  
Mueller Hinton ◽  
Envelope Stress ◽  
Mueller Hinton Agar

ABSTRACTMutations inliaFSR, a three-component regulatory system controlling cell-envelope stress response, were recently linked with the emergence of daptomycin (DAP) resistance in enterococci. Our previous work showed that aliaFmutation increased the DAP MIC of a vancomycin-resistantEnterococcus faecalisstrain from 1 to 3 μg/ml (the DAP breakpoint is 4 μg/ml), suggesting that mutations in theliaFSRsystem could be a pivotal initial event in the development of DAP resistance. With the hypothesis that clinical enterococcal isolates with DAP MICs between 3 and 4 μg/ml might harbor mutations inliaFSR, we studied 38Enterococcus faeciumbloodstream isolates, of which 8 had DAP MICs between 3 and 4 μg/ml by Etest in Mueller-Hinton agar. Interestingly, 6 of these 8 isolates had predicted amino acid changes in the LiaFSR system. Moreover, we previously showed that among 6 DAP-resistantE. faeciumisolates (MICs of >4 μg/ml), 5 had mutations inliaFSR. In contrast, none of 16E. faeciumisolates with a DAP MIC of ≤2 μg/ml harbored mutations in this system (P< 0.0001). All but one isolate withliaFSRchanges exhibited DAP MICs of ≥16 μg/ml by Etest using brain heart infusion agar (BHIA), a medium that better supports enterococcal growth. Our findings provide a strong association between DAP MICs within the upper susceptibility range and mutations in theliaFSRsystem. Concomitant susceptibility testing on BHIA may be useful for identifying theseE. faeciumfirst-step mutants. Our results also suggest that the current DAP breakpoint forE. faeciummay need to be reevaluated.

scholarly journals OptimizedIn VitroAntibiotic Susceptibility Testing Method for Small-Colony Variant Staphylococcus aureus

2016 ◽  
Vol 60 (3) ◽  
pp. 1725-1735 ◽  
Author(s):  
Mimi R. Precit ◽  
Daniel J. Wolter ◽  
Adam Griffith ◽  
Julia Emerson ◽  
Jane L. Burns ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Susceptibility Testing ◽  
Brain Heart Infusion ◽  
Chronic Infections ◽  
Poor Growth ◽  
Content Type ◽  
Treatment History ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
Small Colony

Staphylococcus aureussmall-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST ofS. aureusSCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86S. aureusSCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin;mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.

scholarly journals Simple Phenotypic Tests To Improve Accuracy in Screening Chromosomal and Plasmid-Mediated Colistin Resistance in Gram-Negative Bacilli

2020 ◽  
Vol 59 (1) ◽  
pp. e01701-20
Author(s):  
Fernando Pasteran ◽  
Diego Danze ◽  
Alejandra Menocal ◽  
Carla Cabrera ◽  
Ignacio Castillo ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Agar Plate ◽  
Systematic Screening ◽  
Gram Negative ◽  
Content Type ◽  
Mueller Hinton ◽  
Improve Accuracy ◽  
Mueller Hinton Agar ◽  
Phenotypic Tests ◽  
Mueller Hinton Broth

ABSTRACTCLSI and EUCAST recommend that only broth microdilution (BMD) should be used for routine colistin susceptibility testing; however, this technique can be difficult to perform in resource-poor settings. The purpose of this study was to evaluate the accuracy of a colistin agar spot test (COL-AS) and a colistin drop test (COL-DT) compared to BMD. COL-AS and COL-DT were assessed with a collection of 271 Gram-negative bacilli clinical isolates: 195 Enterobacterales (including 63 mcr-1 positive strains), 37 Acinetobacter spp., and 39 Pseudomonas aeruginosa. For COL-AS, 3.0 μg/ml (final concentration) of colistin was added to a Mueller-Hinton agar plate and subsequently swabbed with a 0.5 McFarland standard suspension of the tested strain within a 1 cm2 spot. For COL-DT, 10 μl of a 16 μg/ml colistin solution was dripped on the surface of a Mueller-Hinton agar plate, previously inoculated with a lawn of the tested strain (0.5 McFarland standard). Colistin solution was made either by dissolving powder or by disk elution in cation-adjusted Mueller-Hinton broth (CA-MHB). Overall, 141/271 (52%) isolates were categorized as colistin resistant by reference BMD. COL-AS yielded a categorical agreement (CA) of 95.5% compared to BMD, with 0.7% very major errors and 3.8% major errors. COL-DT yielded a CA of 96.2% compared to BMD, with 0.7% and 0% very major errors and 3.1% and 3.8% major errors, for colistin powder and disk elution solutions, respectively. Most major errors occurred for mcr-1 strains with MICs that fluctuated from 2 to 4 μg/ml according to the method used. In conclusion, we developed and validated methods suited to the systematic screening of resistance to colistin in Gram-negative bacilli.

scholarly journals Multicenter Evaluation of BioFire FilmArray Meningitis/Encephalitis Panel for Detection of Bacteria, Viruses, and Yeast in Cerebrospinal Fluid Specimens

2016 ◽  
Vol 54 (9) ◽  
pp. 2251-2261 ◽  
Author(s):  
Amy L. Leber ◽  
Kathy Everhart ◽  
Joan-Miquel Balada-Llasat ◽  
Jillian Cullison ◽  
Judy Daly ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
False Negative ◽  
Human Herpesvirus ◽  
Rapid Test ◽  
Complete Analysis ◽  
Specific Test ◽  
Content Type ◽  
Herpesvirus Type ◽  
Varicella Zoster

Rapid diagnosis and treatment of infectious meningitis and encephalitis are critical to minimize morbidity and mortality. Comprehensive testing of cerebrospinal fluid (CSF) often includes Gram stain, culture, antigen detection, and molecular methods, paired with chemical and cellular analyses. These methods may lack sensitivity or specificity, can take several days, and require significant volume for complete analysis. The FilmArray Meningitis/Encephalitis (ME) Panel is a multiplexedin vitrodiagnostic test for the simultaneous, rapid (∼1-h) detection of 14 pathogens directly from CSF specimens:Escherichia coliK1,Haemophilus influenzae,Listeria monocytogenes,Neisseria meningitidis,Streptococcus pneumoniae,Streptococcus agalactiae, cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, varicella-zoster virus, andCryptococcus neoformans/Cryptococcus gattii. We describe a multicenter evaluation of 1,560 prospectively collected CSF specimens with performance compared to culture (bacterial analytes) and PCR (all other analytes). The FilmArray ME Panel demonstrated a sensitivity or positive percentage of agreement of 100% for 9 of 14 analytes. Enterovirus and human herpesvirus type 6 had agreements of 95.7% and 85.7%, andL. monocytogenesandN. meningitidiswere not observed in the study. ForS. agalactiae, there was a single false-positive and false-negative result each, for a sensitivity and specificity of 0 and 99.9%, respectively. The specificity or negative percentage of agreement was 99.2% or greater for all other analytes. The FilmArray ME Panel is a sensitive and specific test to aid in diagnosis of ME. With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship are anticipated.

scholarly journals Applicability and performance of EUCAST’s rapid antimicrobial susceptibility testing (RAST) on primarily sterile body fluids in blood culture bottles in laboratory routine with total lab automation

2021 ◽  
Author(s):  
Jasmin Kaur Jasuja ◽  
Stefan Zimmermann ◽  
Irene Burckhardt
Keyword(s):  
Blood Culture ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Body Fluids ◽  
E Coli ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
And Performance ◽  
Sterile Body Fluids ◽  
Better Than

AbstractOptimisation of microbiological diagnostics in primarily sterile body fluids is required. Our objective was to apply EUCAST’s RAST on primarily sterile body fluids in blood culture bottles with total lab automation (TLA) and to compare results to our reference method Vitek2 in order to report susceptibility results earlier. Positive blood culture bottles (BACTEC™ Aerobic/Anaerobic/PEDS) inoculated with primarily sterile body fluids were semi-automatically subcultured onto Columbia 5% SB agar, chocolate agar, MacConkey agar, Schaedler/KV agar and Mueller-Hinton agar. On latter, cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin were added. After 6 h, subcultures and RAST were imaged and MALDI-TOF MS was performed. Zone sizes were digitally measured and interpreted following RAST breakpoints for blood cultures. MIC values were determined using Vitek2 panels. During a 1-year period, 197 Staphylococcus aureus, 91 Enterococcus spp., 38 Escherichia coli, 11 Klebsiella pneumoniae and 8 Pseudomonas aeruginosa were found. Categorical agreement between RAST and MIC was 96.5%. Comparison showed no very major errors, 2/7 (28.6%) and 1/7 (14.3%) of major errors for P. aeruginosa and meropenem and ciprofloxacin, 1/9 (11.1%) for K. pneumoniae and ciprofloxacin, 4/69 (7.0%) and 3/43 (5.8%) for Enterococcus spp. and vancomycin and ampicillin, respectively. Minor errors for P. aeruginosa and meropenem (1/8; 12.8%) and for E. coli and ciprofloxacin (2/29; 6.5%) were found. 30/550 RAST measurements were within area of technical uncertainty. RAST is applicable and performs well for primarily sterile body fluids in blood culture bottles, partially better than blood-based RAST. Official EUCAST evaluation is needed.

scholarly journals 1455. Potential Mechanisms of Cefiderocol MIC Increase in Enterobacterales in In Vitro Resistance Acquisition Studies

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S730-S730
Author(s):  
Yoshinori Yamano ◽  
Rio Nakamura ◽  
Miki Takemura ◽  
Roger Echols
Keyword(s):  
Iron Transport ◽  
Resistance Mechanisms ◽  
Altered Expression ◽  
Carbapenem Resistant ◽  
Mueller Hinton ◽  
Two Component ◽  
Mueller Hinton Agar ◽  
Related Proteins

Abstract Background Cefiderocol (CFDC) is a novel siderophore, iron-chelating cephalosporin, which is transported into bacteria via iron transporters. CFDC has potent in vitro and in vivo activity against all aerobic Gram-negative bacteria, including carbapenem-resistant strains. To date, clinical isolates with cefiderocol MIC &gt;4 µg/mL have been found infrequently, in which the presence of a few β-lactamases or altered iron transport was found. We investigated potential new mechanisms causing CFDC MIC increases in non-clinical studies. Methods The mutation positions were determined by whole genome sequencing using four K. pneumoniae mutants including two KPC producers and one NDM producer that had shown CFDC MIC increases in previous in vitro resistance-acquisition studies. The mutant strains were obtained at the frequency of 10-7 to &lt; 10-8 by spreading bacteria on standard Mueller‒Hinton agar medium containing CFDC at concentrations of 10× MIC, with or without apo-transferrin (20 μg/mL). CFDC MIC was determined by broth microdilution using iron-depleted cation-adjusted Mueller-Hinton broth based on Clinical and Laboratory Standards Institute guidelines. The emergence of MIC increase mutants was also assessed by in vitro chemostat models under humanized plasma pharmacokinetic exposures of CFDC. Results The possible resistance mechanisms were investigated. Mutation of baeS or envZ, sensors of two-component regulation systems, were found in three or two mutants among the tested four isolates, respectively, and caused the MIC to increase by 4–32-fold. The altered expression level of specific genes by the baeS or envZ mutation could affect CFDC susceptibility, but the specific genes have not been identified. In addition, the mutation of exbD, an accessory protein related to iron transport, was identified in one case and caused the MIC to increase by &gt;8-fold. In vitro chemostat studies using two isolates (one NDM producer and one KPC producer) showed no resistance acquisition during 24-hour exposure. Table. Overview of mutation emergence in five isolates of K. pneumoniae Conclusion The mutation of two-component regulation systems (BaeSR and OmpR/EnvZ) and iron transport-related proteins were shown to be possible mechanisms causing CFDC MIC increases, but these mutants did not appear under human exposures. Disclosures Yoshinori Yamano, PhD, Shionogi & Co., Ltd. (Employee) Rio Nakamura, BSc, Shionogi & Co., Ltd. (Employee) Miki Takemura, MSc, Shionogi & Co., Ltd. (Employee) Roger Echols, MD, Shionogi Inc. (Consultant)

scholarly journals 1457. Serial Passage of Enterobacteriaceae to Explore Development of Carbapenem Resistance

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S731-S731
Author(s):  
Yosef Nissim ◽  
Douglas Slain ◽  
P Rocco LaSala
Keyword(s):  
Susceptibility Testing ◽  
Klebsiella Oxytoca ◽  
Carbapenem Resistance ◽  
Serial Passage ◽  
Resistant Isolate ◽  
Mueller Hinton ◽  
The Us ◽  
Zones Of Inhibition ◽  
Mueller Hinton Agar ◽  
The Impact

Abstract Background Carbapenems are broad-spectrum antibacterials that have seen increased usage for the Enterobacteriales family in recent years. While carbapenem usage has been associated with increased antibacterial resistance, there is currently a lack of data comparing the risk of reduced susceptibility selection by the two most commonly used carbapenems in the US, ertapenem (ERT) and meropenem (MER). We conducted a novel serial passage experiment with clinical isolates of Enterobacteriales to assess the impact of repeated exposure to ERT or MER on phenotypic susceptibility patterns. Methods Non-duplicate clinical Enterobacteriales isolates were selected randomly for inclusion. Antimicrobial susceptibility testing was performed by CLSI disc diffusion methods. Standardized suspensions of isolates were plated on Mueller-Hinton agar, and ERT (10mcg) and MER (10mcg) discs applied. Zones of inhibition were measured and recorded after 16-18 hours incubation. Growth from the innermost zone of inhibition around each disc was used to prepare subsequent suspensions for serial susceptibility testing. This process would be repeated daily for 10 days. Each subsequent serially-passaged isolated was tested against both ERT and MER. Daily zones of inhibition were measured and interpreted. Baseline & final susceptibilities were determined by automated methods (Vitek 2). Results Seventeen Enterobacteriaceae isolates were selected, including: Klebsiella pneumoniae (n=11), Klebsiella oxytoca (n=2), Escherichia coli (n=1), Morganella morganii (n=1), and Enterobacter cloacae (n=2). Despite a greater degree of reductions in zones of inhibition with repeated ERT exposure (vs MER), the overall 10 day trends were not found to be significant different (P=0.529). Resistance developed to ERT in six isolates compared to one MER-resistant isolate (P = 0.053). E. cloacae was the only species to show a significant change between drugs (P=0.010). Two of three isolates that developed reduced zone changes &gt; 10mm to MER were initially exposed to ERT on an earlier plate. Conclusion This novel experiment identified the development of some nonsignificant reductions in susceptibility with ERT after serial exposure. Results from this pilot study should encourage larger well-designed studies in this area. Disclosures All Authors: No reported disclosures

scholarly journals Evaluation of the Carba NP Test for Rapid Detection of Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

2013 ◽  
Vol 57 (9) ◽  
pp. 4578-4580 ◽  
Author(s):  
Nathalie Tijet ◽  
David Boyd ◽  
Samir N. Patel ◽  
Michael R. Mulvey ◽  
Roberto G. Melano
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Positive Predictive Value ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Rapid Detection ◽  
False Negative ◽  
Bacterial Extract ◽  
Content Type ◽  
Negative Results ◽  
False Negative Results

ABSTRACTThe Carba NP test was evaluated against a panel of 244 carbapenemase- and non-carbapenemase-producingEnterobacteriaceaeandPseudomonas aeruginosaisolates. We confirmed the 100% specificity and positive predictive value of the test, but the sensitivity and negative predictive value were 72.5% and 69.2%, respectively, and increased to 80% and 77.3%, respectively, using a more concentrated bacterial extract. False-negative results were associated with mucoid strains or linked to enzymes with low carbapenemase activity, particularly OXA-48-like, which has emerged globally in enterobacteria.

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