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

Abstract

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...

Comparative Evaluation of Four Phenotypic Tests for Detection of Carbapenemase-Producing Gram-Negative Bacteria

“…At least two reasons might have accounted for those differences. First, the distribution of carbapenemase types among CPE isolates comprised a larger proportion of OXA-48 producers (62.2%) that are known to be less well detected by these two assays (8,10,12,14). However, this high proportion of OXA-48 producers is in line with the current epidemiology of CPE observed in several European countries such as Belgium, France, Spain, or Germany (4).…”

“…The Neo-Rapid Carb kit and the Rapidec Carba NP test have been evaluated by several groups (8)(9)(10)(11)(12)(13)(14)(15)(16), but, to our knowledge, no comparison with the novel ␤ Carba test has been published yet. Evaluations of the colorimetric carbapenem hydrolysis assays have mostly focused on Enterobacteriaceae, while reports dealing with nonfermentative Gram-negative bacilli are scarce.…”

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

“…Nordmann and coworkers described a new inhibitor-based biochemical assay for carbapenemase detection, the Carba NP test, which has been developed in two versions; the Carba NP-I assay provides a positive or negative result (carbapenemase detected or not detected), whereas the Carba NP-II test is designed to discriminate among carbapenemase classes A, B, and D (17,18). Two commercially available tests use the principle of imipenem hydrolysis and a colorimetric pH indicator to detect the presence of carbapenemases, i.e., the Rapid Carb Screen kit (Rosco Diagnostica, Taastrup, Denmark) and the Rapidec Carba NP test, which was derived from the Carba NP-I assay and was introduced recently into the market (19,20). The Rapid Carb Screen kit contains three separate vials, whereas the Rapidec Carba NP is performed on one plastic strip with preformed wells, which limits handling and minimizes the potential for confusion of the vials.…”

Multicenter Performance Assessment of Carba NP Test

“…In principle, the test is similar to the classic ␤-lactamase detection assays described by Escamilla and by Skinner and Wise in the 1970s, in which benzylpenicillin acidimetrically produced a red color after hydrolysis by Haemophilus influenzae ␤-lactamase (1, 2). With the Carba NP test, hydrolysis of imipenem produces acid and drives the pH indicator, phenol red, from red to yellow.The Carba NP test has been studied by several investigators (3-14), including evaluations and comparisons to a commercial version (15)(16)(17)(18)(19)(20). However, the literature lacks consistency in the Carba NP methodology.…”

“…The Carba NP test has been studied by several investigators (3-14), including evaluations and comparisons to a commercial version (15)(16)(17)(18)(19)(20). However, the literature lacks consistency in the Carba NP methodology.…”