Evaluation of the Carba NP test for carbapenemase detection in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., and its practical use in the routine work of a national reference laboratory for susceptibility testing

The aim of this study was to evaluate the Carba NP test (and CarbAcineto) for the detection of carbapenemases in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., and to assess its usefulness in the routine work of the National Reference Centre for Susceptibility Testing (NRCST) in Poland. The evaluation of the Carba NP/CarbAcineto tests was carried out on a group of 81 Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. isolates producing KPC-, NDM-, VIM-, IMP- or OXA-48, -23, -24/40, -58-type carbapenemases, and on 26 carbapenemase-negative strains cultivated on a broad panel of microbiological media. Subsequently, the performance of the Carba NP/CarbAcineto tests was assessed on 1282 isolates of Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. from Polish hospitals, submitted to the NRCST during a 9-month period in 2014. The Carba NP/CarbAcineto results were compared with other phenotypic tests and/or polymerase chain reaction (PCR). The impact of the media on the results of the Carba NP/CarbAcineto tests was observed, with the Columbia blood agar yielding the highest sensitivity and clarity of the results. Furthermore, the Carba NP/CarbAcineto tests were included in the NRCST routine procedure for carbapenemase identification. The sensitivity and specificity of the Carba NP test were 95.8% and 93.3%, respectively, for Enterobacteriaceae, and 97.5% and 99.0%, respectively, for Pseudomonas spp. The sensitivity of the CarbAcineto test for Acinetobacter spp. was 88.9%. This study confirmed the usefulness of the Carba NP/CarbAcineto tests for the rapid detection of various types of carbapenemases.

The aim of this study was to evaluate the Carba NP and CarbAcineto tests for the detection of carbapenemaseproducing Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. cultivated on different media. Moreover, the Carba NP/CarbAcineto tests were assessed in the routine diagnostics of carbapenemase producers conducted by the National Reference Centre for Susceptibility Testing (NRCST) in Poland in 2014.

Materials and methods
Assessment of the Carba NP and CarbAcineto tests: strain collection and cultivation media For the initial evaluation of the Carba NP and CarbAcineto tests, a total of 107 strains were used: 81 carbapenemase producers that were well characterised with phenotypic methods, polymerase chain reaction (PCR) and sequencing, and 26 carbapenemase non-producers, including 21 meropenemnon-susceptible strains (Table 1). Both tests were performed by the in-house protocol as previously described [14,18]. The results presented are those that were recorded after 2 h of incubation time. For cultivation of bacteria, a number of non-selective or screening media and the Mueller-Hinton ready-to-use agar plates, products of six manufacturers, were used (Tables 2 and 3).

The Carba NP and CarbAcineto tests in routine reference diagnostics
In the second part of this work, the Carba NP and CarbAcineto tests were implemented into the routine activity of the NRCST, which is responsible for the reference diagnostics and surveillance of carbapenemase-producing Gram-negative pathogens in Poland. Both tests were performed along with the phenylboronic acid disc test [27], double-disc synergy test with EDTA (DDST-EDTA) [28], temocillin disc test [29] for the phenotypic detection of KPCs, MBLs and OXA-48 types, respectively [30], spectrophotometric assay for imipenem hydrolysis [31] and PCR for bla KPC -, bla VIM -, bla IMP -, bla NDM -, bla OXA-48 -, bla OXA-23 -, bla OXA-24/40 -and bla OXA-58 -like genes [32]. This analysis was done on 1282 isolates in total (Table 4), including 915 Enterobacteriaceae, 309 Pseudomonas spp. and 58 Acinetobacter spp. collected from April to the end of December 2014 in hospitals all over Poland. All these isolates were cultivated only on Columbia blood agar plates.

Assessment of the Carba NP and CarbAcineto tests and the cultivation media
For Enterobacteriaceae, the Carba NP test performed well, with the carbapenemase-producing strains cultured on most of the non-selective or screening media ( Table 2).
Falsenegative results were obtained only for single isolates with VIMs or OXA-48s grown on three types of chromogenic media. When bacteria were cultivated on Mueller-Hinton (MH) agars, the Carba NP test was positive for all KPC, IMP and all but one OXA-48 producers, while false-negative or questionable results were obtained for NDM-and VIMproducing strains. For a number of carbapenemase-negative strains picked up from some media, the results were questionable and for single strains were false-positive.
The results of the Carba NP test for Pseudomonas spp. strains are presented in Table 3. In the case of carbapenemase (VIM or IMP) producers, the best results were observed for  Table 3. As in the case of Enterobacteriaceae and Pseudomonas spp., the impact of the cultivation medium on CarbAcineto was revealed; moreover, for isolates with OXA-like carbapenemases, the correct results were observed more often than for VIM-positive strains ( Table 3).

Discussion
The Carba NP test was described for the first time in 2012 by Nordmann et al. as a rapid, easy and cheap method for carbapenemase detection in Enterobacteriaceae and Pseudomonas spp., with high sensitivity and specificity [1,15]. In previous studies, the test was evaluated by several groups, often confirming the original observations [2,19,[33][34][35]. However, one study undermined the sensitivity in the case of MBL-producing strains [2]. The problem turned out to be associated with the cultivation medium used [16][17][18], and the follow-up study by Dortet et al. documented well the impact of microbiological media on the performance of Carba NP [18].
In the first part of this work, well-characterised Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. strains with various carbapenemases were used, along with a broad panel of commercially available media. The results clearly illustrated the influence of the media on the Carba NP/CarbAcineto results. According to our experience, the Columbia blood agar is the best choice for the cultivation of bacteria for the Carba NP/CarbAcineto tests, providing the highest sensitivity and clarity of the results. The VIM or NDM producers harvested from most of the MH agars yielded a remarkable number of false-negative results, which were most probably related to lower zinc ions' concentrations that is crucial for the MBL activity [18]. Owing to the low incidence of OXA-48 producers in Poland so far, only six such isolates were used in this work, and all of these were clearly positive in Carba NP, in contrast to some other reports [17,25,[34][35][36][37]. This study has definitely confirmed the usefulness of the CarbAcineto test for the detection of the acquired carbapenemases in Acinetobacter spp. [8,38,39] and for distinguishing such isolates from hyperproducers of the natural OXA-51 types and from the carbapenemase-negative carbapenem-non-susceptible isolates, which is important for epidemiological reasons.
The Carba NP and CarbAcineto tests have been used in the NRCST from the end of 2013 and March 2014, respectively, and now are included in its routine algorithm for carbapenemase detection as reliable, inexpensive and easy  NPV 99.0% (95% CI, 97.6-100%) a PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval screening tests, reducing remarkably the time for the first feedback information for clinical laboratories. Based on own experience, and in the context of the alarming spread of carbapenemase-producing Gram-negative pathogens in Poland, the NRCST has been recommending Carba NP/ CarbAcineto tests for use in diagnostic microbiology laboratories all over the country.