Rapid and Simultaneous Detection of
 bla
 KPC
 and
 bla
 NDM
 by Use of Multiplex Real-Time PCR

Milillo, Michael; Kwak, Yoon I.; Snesrud, Erik; Waterman, Paige; Lesho, Emil; McGann, Patrick

doi:10.1128/jcm.03316-12

Cited by 30 publications

(18 citation statements)

References 21 publications

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“…The high level of accuracy of the Carba NP test in this study was consistent with previous reports (8,9,(12)(13)(14)(15). The achievement of almost comparable sensitivity with the Rosco test was due to enhancing the clarity of test interpretation with a modified approach to reading what were otherwise uninterpretable tests.…”

Section: Discussionsupporting

confidence: 80%

Comparison of the Carba NP, Modified Carba NP, and Updated Rosco Neo-Rapid Carb Kit Tests for Carbapenemase Detection

et al. 2015

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The accurate detection of carbapenemase-producing organisms is a major challenge for clinical laboratories. The Carba NP test is highly accurate but inconvenient, as it requires frequent preparation of fresh imipenem solution. The current study was designed to compare the Carba NP test to two alternative tests for accuracy and convenience. These were a modified Carba NP test that utilized intravenous (i.v.) imipenem-cilastatin, which is less expensive than reference standard imipenem powder, and an updated version of the Rosco Neo-Rapid Carb kit, which does not require the preparation of imipenem solution and has a shelf life of 2 years. The comparison included 87 isolates that produced class A carbapenemases (including KPC-2, -3, -4, -5, -6, and -8, NMC-A, and SME type), 40 isolates that produced metallo-␤-lactamases (including NDM-1, GIM-1, SPM-1, IMP-1, -2, -7, -8, -18, and -27, and VIM-1, -2, and -7), 11 isolates that produced OXA-48, and one isolate that produced OXA-181. Negative controls consisted of 50 isolates that produced extended-spectrum ␤-lactamases (ESBLs), AmpCs (including hyperproducers), K1, other limited-spectrum ␤-lactamases, and porin and efflux mutants. Each test exhibited 100% specificity and high sensitivity (Carba NP, 100%; Rosco, 99% using modified interpretation guidelines; and modified Carba NP, 96%). A modified approach to interpretation of the Rosco test was necessary to achieve the sensitivity of 99%. If the accuracy of the modified interpretation is confirmed, the Rosco test is an accurate and more convenient alternative to the Carba NP test.T he accurate detection of carbapenemase-producing organisms (CPOs) is a major challenge for clinical laboratories. In some laboratories, detection of carbapenem-resistant Enterobacteriaceae (CRE) is the primary focus, and the need to detect carbapenemase production is considered optional and for epidemiologic purposes only (1). CRE detection is based on detection of resistance or nonsusceptibility of Enterobacteriaceae to carbapenems and certain cephalosporins and does not distinguish between carbapenemase producers and non-carbapenemase producers. Although carbapenem-resistant non-carbapenemase producers are important, they should not trigger the same level of concern as CPOs such as NDM-and other carbapenemase-producing isolates (2-6). Furthermore, the focus on CRE detection ignores carbapenemase producers that either are not Enterobacteriaceae or are carbapenemase-producing Enterobacteriaceae that are carbapenem susceptible, such as the VIM-producing Klebsiella pneumoniae isolates with imipenem MICs as low as 0.12 g/ml that were involved in a large outbreak with high mortality in Greece (7).Clinical laboratories aiming to detect carbapenemase producers need a test that is accurate and convenient. The Carba NP test is highly accurate (8, 9) but labor-intensive and inconvenient due to the instability of imipenem in solution, which necessitates extemporaneous preparation (1, 10). Another disadvantage of this test is the high cost of reference...

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Section: Discussionsupporting

confidence: 80%

Comparison of the Carba NP, Modified Carba NP, and Updated Rosco Neo-Rapid Carb Kit Tests for Carbapenemase Detection

et al. 2015

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“…False-negative Carba NP results were reported previously, e.g., for Providencia rettgeri, Providencia stuartii, or Proteus mirabilis, in particular for mucoid isolates and isolates with NDM enzymes (11,29,31). Negative results were linked to variations in protein extraction, to species-specific characteristics, or to the agar type and ion content used for cultivation of the isolates in question.…”

Section: Discussionmentioning

confidence: 91%

Evaluation of the Rapidec Carba NP Test for Detection of Carbapenemases in Enterobacteriaceae

Hombach

Gunten²,

Castelberg

et al. 2015

J Clin Microbiol

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bThis study evaluated the performance of the Rapidec Carba NP test, which was introduced recently into the market for the detection of carbapenemase production in a broad spectrum of ␤-lactamase-producing Enterobacteriaceae clinical isolates. In total, 252 clinical Enterobacteriaceae isolates that had been genetically characterized with respect to carbapenemase, extended-spectrum ␤-lactamase (ESBL), and AmpC genes were analyzed; 51/252 isolates (20.2%) were genetically confirmed to be carbapenemase producers, whereas 201/252 isolates (79.8%) were genetically negative for the presence of carbapenemase genes. The Rapidec Carba NP test was applied according to the manufacturer's instructions, and results were read after 30 and 120 min of incubation. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Rapidec Carba NP test were 90.2%, 100%, 100%, and 97.6%, respectively, when the manufacturer's instructions were followed. Four of 5 false-negative results occurred with OXA-48-like enzymes. After an incubation time of 30 min, the sensitivity was 49%. The sensitivity increased to 100% when the recommended bacterial inoculum was doubled and the test was read strictly after 120 min of incubation. The Rapidec Carba NP test is a useful tool for the reliable confirmation of carbapenemase-producing Enterobacteriaceae isolates. The test should be read strictly after 120 min of incubation and the inoculum should be larger than recommended by the manufacturer.

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“…1). False-negative Carba NP results have also been described for mucoid colonies, e.g., of P. rettgeri, P. stuartii, or P. mirabilis isolates (29,31). Negative results were attributed to difficulties in protein extraction, species-specific traits, or the influence of the agar type and ion content of the Carba NP test (30,31,36).…”

Section: Discussionmentioning

confidence: 99%

“…In 2014, EUCAST published new guidelines for the detection of resistance mechanisms, including carbapenemases, in which a CDT is recommended for carbapenemase confirmation (14,22,25). Recently, Nordmann et al described a new inhibitor-based biochemical assay for carbapenemase detection, the Carba NP test, which has been published in two versions: the Carba NP-I assay provides a positive or negative result (carbapenemase detected/not detected), whereas the Carba NP-II test has been designed to also discriminate among carbapenemase classes A, B, and D (26)(27)(28)(29). Apart from the original publications, few studies have systematically evaluated the Carba NP-I test for Enterobacteriaceae, and both the originally published protocol and modified versions were used.…”

mentioning

confidence: 99%

Evaluation of Carbapenemase Screening and Confirmation Tests with Enterobacteriaceae and Development of a Practical Diagnostic Algorithm

et al. 2015

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Reliable identification of carbapenemase-producing members of the family Enterobacteriaceae is necessary to limit their spread. This study aimed to develop a diagnostic flow chart using phenotypic screening and confirmation tests that is suitable for implementation in different types of clinical laboratories. A total of 334 clinical Enterobacteriaceae isolates genetically characterized with respect to carbapenemase, extended-spectrum ␤-lactamase (ESBL), and AmpC genes were analyzed. A total of 142/334 isolates (42.2%) were suspected of carbapenemase production, i.e., intermediate or resistant to ertapenem (ETP) and/or meropenem (MEM) and/or imipenem (IPM) according to EUCAST clinical breakpoints (CBPs). A group of 193/334 isolates (57.8%) showing susceptibility to ETP, MEM, and IPM was considered the negative-control group in this study. CLSI and EUCAST carbapenem CBPs and the new EUCAST MEM screening cutoff were evaluated as screening parameters. ETP, MEM, and IPM with or without aminophenylboronic acid (APBA) or EDTA combined-disk tests (CDTs) and the Carba NP-II test were evaluated as confirmation assays. EUCAST temocillin cutoffs were evaluated for OXA-48 detection. The EUCAST MEM screening cutoff (<25 mm) showed a sensitivity of 100%. The ETP APBA CDT on Mueller-Hinton agar containing cloxacillin (MH-CLX) displayed 100% sensitivity and specificity for class A carbapenemase confirmation. ETP and MEM EDTA CDTs showed 100% sensitivity and specificity for class B carbapenemases. Temocillin zone diameters/MIC testing on MH-CLX was highly specific for OXA-48 producers. The overall sensitivity, specificity, positive predictive value, and negative predictive value of the Carba NP-II test were 78.9, 100, 100, and 98.7%, respectively. Combining the EUCAST MEM carbapenemase screening cutoff (<25 mm), ETP (or MEM), APBA, and EDTA CDTs, and temocillin disk diffusion on MH-CLX promises excellent performance for carbapenemase detection. In recent years, the emergence of diverse carbapenemases in members of the family Enterobacteriaceae has become a major challenge for health care systems (1). Carbapenemase-producing bacterial isolates pose a severe clinical problem, as nonsusceptibility to beta-lactams is frequently accompanied by coresistance to additional drug classes, e.g., aminoglycosides or quinolones (2, 3). As a consequence, treatment options for carbapenemase producers are alarmingly limited and often drugs displaying significant side effects need to be administered as a last resort (4).␤-Lactamases are classified according to their functional properties and molecular structure by Ambler and Bush (5, 6). Some of these enzymes also display hydrolytic activity toward carbapenems, e.g., Klebsiella pneumoniae carbapenemase (KPC, Ambler/ Bush class A)-, the New Delhi metallo-␤-lactamase (NDM-1)-, VIM-, and GIM-type enzymes (all Ambler/Bush class B) or OXA-48 (Ambler/Bush class D). A key characteristic used to discriminate enzymes belonging to different Ambler/Bush classes is responsiveness to specific inhibitors: class...

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Rapid and Simultaneous Detection of bla _KPC and bla _NDM by Use of Multiplex Real-Time PCR

Cited by 30 publications

References 21 publications

Comparison of the Carba NP, Modified Carba NP, and Updated Rosco Neo-Rapid Carb Kit Tests for Carbapenemase Detection

Comparison of the Carba NP, Modified Carba NP, and Updated Rosco Neo-Rapid Carb Kit Tests for Carbapenemase Detection

Evaluation of the Rapidec Carba NP Test for Detection of Carbapenemases in Enterobacteriaceae

Evaluation of Carbapenemase Screening and Confirmation Tests with Enterobacteriaceae and Development of a Practical Diagnostic Algorithm

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Rapid and Simultaneous Detection of bla KPC and bla NDM by Use of Multiplex Real-Time PCR

Cited by 30 publications

References 21 publications

Comparison of the Carba NP, Modified Carba NP, and Updated Rosco Neo-Rapid Carb Kit Tests for Carbapenemase Detection

Comparison of the Carba NP, Modified Carba NP, and Updated Rosco Neo-Rapid Carb Kit Tests for Carbapenemase Detection

Evaluation of the Rapidec Carba NP Test for Detection of Carbapenemases in Enterobacteriaceae

Evaluation of Carbapenemase Screening and Confirmation Tests with Enterobacteriaceae and Development of a Practical Diagnostic Algorithm

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Product

Resources

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Rapid and Simultaneous Detection of bla _KPC and bla _NDM by Use of Multiplex Real-Time PCR