Current Trends in Culture-Based and Molecular Detection of Extended-Spectrum-β-Lactamase-Harboring and Carbapenem-Resistant Enterobacteriaceae

Gazin, Muriel; Paasch, Fabienne; Goossens, Herman; Malhotra-Kumar, Surbhi

doi:10.1128/jcm.06852-11

Cited by 110 publications

(90 citation statements)

References 27 publications

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“…However, as by now there are no reasonable molecular solutions (16), this screening strategy might be the best option to maximize the sensitivity of the screening process and might therefore improve the empirical treatment of colonized patients and the prevention of the nosocomial spread of ESBL-E.…”

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Use of an Enrichment Broth Improves Detection of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae in Clinical Stool Samples

2016

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cThis study evaluated the impact of preenrichment on the detection of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in clinical stool samples. ESBL-E were detected in 41 of 343 patients (12.0%). As 31.7% of the ESBL-E carriers were identified by preenrichment, only this additional diagnostic step significantly improved the detection of ESBL-E. E xtended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) are increasingly reported worldwide. Treatment options for infections due to these organisms are limited, and initial empirical therapy is often inappropriate and is associated with increased morbidity and mortality (1). ESBL-E carriers are known to develop bacteremia with the isolates colonizing the patients' intestines (2). Therefore, in many hospitals, risk-adapted screening for ESBL-E carriage is performed, but limited data exist on the optimal screening strategy. Currently, laboratory detection of ESBL-E in fecal specimens mostly relies on culture methods using selective agar medium. This method has a modest sensitivity (3). Preenrichment of swabs is used by many laboratories for methicillin-resistant Staphylococcus aureus (MRSA) screening and has demonstrated significantly improved sensitivity (4). To our knowledge, the impact of preenrichment on ESBL-E detection in stool samples has not been systematically investigated. In our study, we compared three different selective and nonselective broths for the detection of ESBL-E in clinical stool samples to the standard procedure-direct plating on ESBL agar.(This study was presented in part as a poster at the 25th European Congress of Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 25 to 28 April 2015.) From September 2013 to March 2014, 496 stool samples submitted for ESBL-E screening from 343 patients at the University Hospital Cologne, a 1,400-bed tertiary care facility, were included in the study. All samples were stored at 4°C and were processed within 24 h of receipt. A pea-sized portion of solid stool or 250 l of liquid stool was suspended in 1 ml 0.9% NaCl to reach a standardized inoculum. One hundred microliters of the stool suspension was inoculated directly onto chromID ESBL agar (bioMérieux, Marcy l'Etoile, France) and into 5 ml of nonselective tryptic soy broth (TSB), MacConkey (MC) broth (Roth, Germany, Karlsruhe) selecting only Gram-negative rods, and MacConkey broth supplemented with cefuroxime (32 mg/liter) and vancomycin (64 mg/liter) (MC-CV) additionally inhibiting the nonresistant Gram-negative flora and further inhibiting Grampositive bacteria. Each medium was stored and processed according to the manufacturers' instructions.Agar plates and broths were incubated at 36°C Ϯ 1°C in ambient air for 18 h to 24 h. Using calibrated inoculation loops, 10 l of the respective enrichment broth was inoculated onto ESBL agar, which was incubated at 36°C Ϯ 1°C in ambient air for another 18 h to 24 h. Plates were interpreted according to the manufacturer's instructions. Uncolored colonies growing on ...

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Use of an Enrichment Broth Improves Detection of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae in Clinical Stool Samples

2016

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“…Those techniques require a preliminary growth step of 24 to 48 h (4). Molecular detection of ESBL-coding genes is interesting but remains costly, requires expertise, and does not detect all genes encoding enzymes exhibiting ESBL activity (4)(5)(6). Other techniques, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (7), are being developed, but they do require additional material and a significant degree of expertise.…”

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Comparison of Three Biochemical Tests for Rapid Detection of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae

2016

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dEnterobacterial isolates producing clavulanic-inhibited extended-spectrum ␤-lactamases (ESBLs) are increasingly spreading in the community and are often responsible for nosocomial infections. Rapid biochemical tests have been developed recently for their detection. Three tests, namely, the Rapid ESBL NDP test, the ␤-Lacta test, and the Rapid ESBL Screen, have been evaluated with a collection of 108 well-characterized strains, including wild-type strains, strains producing ESBLs, overexpressed cephalosporinases, and carbapenemases. The ESBL NDP test and the Rapid ESBL Screen (a copy of the ESBL NDP test) are aimed at detecting ESBL producers, while the ␤-Lacta test is aimed at detecting not only ESBL producers but also cephalosporinase and carbapenemase producers. The sensitivity and specificity for detecting ESBL producers (n ‫؍‬ 60) were 95% and 100% for the Rapid ESBL NDP test, 80% and 87% (after 30 min) and 92% and 83% (after 2 h) for the Rapid ESBL Screen, and 88% and 71% for the ␤-Lacta test, respectively. Varied and time-consuming detection (up to 2 h) of ESBLs by the Rapid ESBL Screen and concomitant and varied detection of producers of AmpC and several types of carbapenemases correspond to significant shortcomings of using the Rapid Screen ESBL and ␤-Lacta tests, respectively.A cquired resistance to broad-spectrum cephalosporins in Enterobacteriaceae is mainly due to the production of clavulanic acid-inhibited extended-spectrum ␤-lactamases (ESBLs), which have extensively disseminated worldwide (1). Along with carbapenemase producers, ESBL producers represent the most important resistance trait in Enterobacteriaceae in 2015. The European Antimicrobial Resistance Surveillance System Network (EARSNet), which includes 30 European countries, reported in 2013 the prevalence rates of nonsusceptibility to broad-spectrum cephalosporins among invasive enterobacterial isolates (2). The proportion of Escherichia coli strains resistant to broad-spectrum cephalosporins ranged from 5% to 39.6%, depending on the country. In Klebsiella pneumoniae, the percentage of resistance to broad-spectrum cephalosporins showed a significant increase from 22.8% in 2012 to 30% in 2013, encompassing 85 to 100% of the ESBL producers. In the United States, the percentages of health care-associated infections caused by broad-spectrum cephalosporin-resistant Enterobacteriaceae have been estimated to be 14% and 23% for E. coli and Klebsiella spp., respectively (http://www.cdc.gov /drugresistance/threat-report-2013). Those ESBL-producing Enterobacteriaceae are identified either as a source of hospital-or community-acquired infections (3).The rapid detection of ESBL producers is therefore crucial in order to prevent their dissemination and to guide the treatments of infected patients. Several phenotypic techniques are based on the inhibition of ESBL activity by clavulanic acid or tazobactam. Those techniques require a preliminary growth step of 24 to 48 h (4). Molecular detection of ESBL-coding genes is interesting but remains costly, requi...

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“…DNA PCR and microarrays are commercially available, but they can be labor-intensive and expensive and may have slow turnaround compared to CNP and RCS (11). Adding to the challenge is that there are currently no FDA-cleared molecular assays that are designed to detect carbapenemase determinants from bacterial colonies on the U.S. market.…”

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Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates

et al. 2015

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fThis study compared the performance of the Carba NP assay, published by the Clinical and Laboratory Standards Institute, and the Rosco Rapid Carb Screen kit. Carba NP had superior sensitivity, but both assays required an increased inoculum to detect carbapenemase production in isolates with bla NDM , bla IMP , and bla OXA-48 . Rapid detection of carbapenemase production in multidrugresistant organisms may help to mitigate institutional outbreaks by expediting initiation of infection control procedures (1). Nordmann et al. first described the Carba NP assay and reported sensitivity and specificity of 100% (2). Carbapenemase activity is detected using a pH indicator (phenol red) that changes color with the hydrolysis of the ␤-lactam ring of imipenem. Dortet et al. (3) published a modified procedure in 2014. The bacterial inoculum was reduced, testing was streamlined using whole lysed bacterial cells instead of supernatant, and the concentration of imipenem was raised from 3 to 6 mg/ml (3). Reported sensitivity and specificity were 100% (3). In 2015, based on the work of Vasoo et al. (4), the Clinical and Laboratory Standards Institute (CLSI) published a similar Carba NP procedure using a bacterial inoculum that was further reduced to 1 l (5).Carba NP is labor-intensive. Reagents need to be prepared inhouse, and some have shelf lives as short as 72 h. In contrast, the Rapid Carb Screen kit (98021; Rosco Diagnostica A/S, Taastrup, Denmark) provides tablets containing a pH indicator, with and without imipenem. No reagent preparation is required, and the shelf life is Ͼ12 months. In this study, we evaluated the performance of the Carba NP assay (CNP) as published by CLSI and the Rapid Carb Screen (RCS).In total, 49 organisms were tested. These included 39 isolates that were previously characterized as carrying bla KPC , bla NDM , bla VIM , bla IMP , and bla . Real-time PCR was performed to detect bla KPC , bla NDM , and bla VIM as described elsewhere (6, 7). bla IMP , and bla OXA-48 were detected using conventional PCR with primers as follows: for bla IMP , 5=-GTTTATGT TCATACWTCG-3= (forward) and 5=-GGTTTAAYAAAACAA CCAC-3= (reverse); for bla OXA-48 , 5=-ATGCGTGTATTAGCCT TATCGGCTG-3= (forward) and 5=-CTAGGGAATAATTTTTT CCTGTTTG-3= (reverse). In addition, we included 10 isolates that tested negative for all targets. Imipenem, meropenem, and ertapenem MICs were produced using GN4F Sensititre Gramnegative MIC plates (Thermo Fisher Scientific, Oakwood Village, OH) and interpreted using CLSI interpretive breakpoints (5). Organisms were identified to the species level using the Vitek 2 system (bioMérieux, Durham, NC). Prior to testing, isolates were subcultured and incubated twice in ambient air at 37°C on Trypticase soy agar with 5% sheep's blood (Remel, Lenexa, KS). All testing runs included positive (ATCC BAA-1705) and negative (ATCC BAA-1706) controls. CNP also had a reagent-only control.CNP was performed strictly as stated by CLSI (3). Briefly, solution A with 0.5% phenol red solution (made with phenol red indicator po...

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Current Trends in Culture-Based and Molecular Detection of Extended-Spectrum-β-Lactamase-Harboring and Carbapenem-Resistant Enterobacteriaceae

Cited by 110 publications

References 27 publications

Use of an Enrichment Broth Improves Detection of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae in Clinical Stool Samples

Use of an Enrichment Broth Improves Detection of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae in Clinical Stool Samples

Comparison of Three Biochemical Tests for Rapid Detection of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae

Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates

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