Bacteriology

Evaluation of the Amplidiag CarbaR+VRE Kit for Accurate Detection of Carbapenemase-Producing Bacteria

Saoussen Oueslati, Delphine Girlich, Laurent Dortet, Thierry Naas
Karen C. Carroll, Editor
DOI: 10.1128/JCM.01092-17

ABSTRACT

As carbapenemase-producing Gram-negative bacilli (CP-GNB) (Enterobacteriaceae, Pseudomonadaceae, and Acinetobacter spp.) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The Amplidiag CarbaR+VRE assay is a multiplex nucleic acid-based in vitro diagnostic test intended for the detection of CP-GNB and vancomycin-resistant enterococci (VRE) from cultured colonies. We have evaluated its ability to detect carbapenemase genes in 100 well-characterized GNB and in 200 consecutive enterobacterial isolates with reduced susceptibility to carbapenems that were referred to the French National Reference Center for carbapenem resistance. The assay has been validated on purified DNA but also directly on colonies. The Amplidiag CarbaR+VRE assay could detect all KPC, NDM, VIM, IMP, and OXA-48-like variants tested and all acquired carbapenem-hydrolyzing oxacillinases from Acinetobacter baumannii (OXA-23, OXA-24/-40, and OXA-58) as well as the overexpressed chromosomally encoded OXA-51-like β-lactamase associated with an upstream inserted ISAba1. However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES-5, and GES-14), GIM-1, AIM-1, SPM-1, DIM-1, OXA-198 in Pseudomonas aeruginosa, or OXA-143-like in A. baumannii were not detected. Amplidiag CarbaR+VRE's performance values were high (100% sensitivity and 99% specificity) as it could detect the five major carbapenemases—NDM, VIM, IMP, KPC, and OXA-48—as well as OXA-type carbapenemases from Acinetobacter spp. that are currently emerging also among Proteus mirabilis and other enterobacterial isolates. It can provide a result directly from colonies growing on Mueller-Hinton (MH) agar or on selective screening medium in less than 2 h. Further evaluations are now necessary to determine the performance values directly on rectal swabs.

INTRODUCTION

Carbapenemase-producing Enterobacteriaceae (CPE) and nonfermenters (CPNF; Pseudomonadaceae and Acinetobacter species) have increasingly been reported worldwide (1, 2). The most clinically relevant carbapenemases belong to either Ambler class A (KPC type); Ambler class B, i.e., metallo-β-lactamases (MBLs) (such as IMP, VIM, and NDM types); or Ambler class D, such as (i) OXA-48-like in Enterobacteriaceae; (ii) OXA-23, OXA-24/-40, OXA-58, OXA-143, and the overexpressed intrinsic OXA-51-like enzymes in Acinetobacter spp.; and (iii) OXA-198 in Pseudomonas aeruginosa (3–6).

There is a large heterogeneity of carbapenem resistance mechanisms depending on the bacterial species and its geographical location. Concerning Enterobacteriaceae, KPC dominates in the United States, Greece, Italy, and Israel (1, 4). The IMP-1 enzyme is mostly prevalent in Japan, and OXA-48-like carbapenemases are of great concern in the Middle East, in most Mediterranean countries, and in many European countries such as France, where a large variety of enzymes are present (7). In Pseudomonas spp. and Acinetobacter spp., Ambler class A, B, and D carbapenemases have been detected. In addition to the acquired OXA carbapenemases (mostly OXA-23, OXA-24/-40, OXA-58, and OXA-143), the Acinetobacter baumannii-Acinetobacter calcoaceticus complex possesses a chromosomally encoded OXA-51-like enzyme with weak carbapenemase activity (6.). However, transposition of an insertion sequence (IS), mostly ISAba1, upstream of the blaOXA-51-like gene can lead to overexpression of the latter, resulting in carbapenem resistance (5). Currently, in the United States and Europe, acquired OXA carbapenemase production (particularly of OXA-23) is the most prevalent mechanism among A. baumannii isolates. At the opposite end, mutations or the loss of expression in OprD porin, even in the absence of any carbapenemase, is the primary mechanism of resistance to imipenem among P. aeruginosa species (5). However, in Pseudomonas spp. the most prevalent carbapenemases worldwide are of the VIM and IMP types (5).

Simple, rapid, and accurate methods are needed to detect all carbapenemase producers and discriminate them among non-carbapenemase producers in order to implement proper infection control measures to prevent further spread in hospitals. Various tests (combined with algorithms) have been developed for the early detection of carbapenemase producers: (i) algorithms based on disc diffusion (8–11); (ii) combination disc diffusion assays (9); (iii) tests able to detect a carbapenem-hydrolyzing activity, such as the Carba NP test and derivatives (12), matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) protocols (13), the BYG test (14), the carbapenem inactivation method (CIM) test (15), and the β-Carba test (16); (iv) immunochromatographic tests for the rapid detection of KPC, OXA-48-type, and NDM carbapenemases (17, 18); and (v) molecular biology-based techniques that all aim to detect the most widespread carbapenemase-encoding genes from bacterial colonies and/or directly from clinical samples (such as rectal swabs) (19, 20).

Molecular diagnostic assays include DNA amplification-based methods: PCR, loop-mediated isothermal amplification (LAMP), microarrays, and whole-genome sequencing (WGS) (19, 21, 22). These methods detect the presence of a carbapenemase gene likely accounting for carbapenem resistance (19). The most common commercially available molecular assays are (i) the Xpert Carba-R kit (Cepheid, Sunnyvale, CA, USA), which detects KPC, NDM, VIM, IMP-1-like, and OXA-48-like carbapenemases; (ii) Eazyplex SuperBug CRE (Amplex Biosystems GmbH, Giessen, Germany), which detects KPC, NDM, VIM, and OXA-48 carbapenemases; (iii) Eazyplex complete A (Amplex Biosystems), which detects also OXA-23, OXA-40, and OXA-58 from Acinetobacter spp.; and (iv) the Check-Direct CPE assay (Check-Points, Wageningen, The Netherlands), which detects KPC, NDM, VIM, and OXA-48. Most of these assays detect at least the four most prevalent carbapenemases, representing more than 95% of the carbapenemases produced by Enterobacteriaceae in France (19, 20, 23).

Here, we have evaluated the performance of Amplidiag CarbaR+VRE (Mobidiag Ltd., Espoo, Finland), a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for the detection of carbapenemase-encoding genes in Enterobacteriaceae and nonfermenting rods as well as vancomycin (Van) resistance related to ligase genes vanA and vanB in enterococci (VRE).

(Part of these results have been presented at ECCMID 2017, Vienna, Austria.)

MATERIALS AND METHODS

Bacterial isolates.A total of 100 collection isolates with characterized β-lactamase content were used in this study. They included 50 Enterobacteriaceae, 30 Pseudomonas species, and 20 Acinetobacter baumannii isolates (see Tables S1, S2, S3, S4, and S5 in the supplemental material). These isolates comprised 79 carbapenemase-producing Gram-negative bacilli (CP-GNB) and 21 non-CP-GNB. The 79 CP-GNB consisted of 41 Enterobacteriaceae (9 KPC, 5 NDM, 4 VIM, 3 IMP, and 15 OXA-48-like producers and 5 isolates producing multiple carbapenemases [three NDM plus OXA-48-like, one NDM-1 plus VIM-2, and one VIM-4 plus OXA-48] [Table S2]), 20 P. aeruginosa isolates (2 KPC-2, 2 GES-like, 7 VIM-like, 4 IMP-like, 1 GIM-1, 1 AIM-1, 1 SPM-1, 1 DIM-1, and 1 OXA-198) (Table S3), and 18 Acinetobacter isolates (4 ISAba1-OXA-51-like, 3 OXA-23, 2 OXA-24/-40, 2 OXA-58, 2 OXA-143-like, 2 NDM, 1 IMP, 1 VIM and 1 GES-14) (Table S4). Non-CP-GNB (n = 21) were 9 Enterobacteriaceae, 10 P. aeruginosa, and 2 A. baumannii isolates. Non-CP-GNB also included 2 OXA-48-like producers that are devoid of carbapenem-hydrolytic activity (OXA-163 and OXA-405) (22–24), which usually give false-positive results with molecular methods (Table S2) (15). A collection of well-characterized Proteus mirabilis isolates have been tested subsequently. These isolates comprised 10 carbapenemase producers (8 OXA-23 and 2 OXA-58) and 2 non-carbapenemase producers.

From 20 January to 10 February 2016, 200 consecutive enterobacterial isolates were referred to the French National Reference Center (F-NRC) for Antibiotic Resistance for evaluation. Reidentification of these 200 isolates revealed 198 Enterobacteriaceae and 2 A. baumannii isolates.

Susceptibility testing.Antimicrobial susceptibilities were determined by the disc diffusion technique on Mueller-Hinton (MH) agar (Bio-Rad, Marnes-La-Coquette, France) and interpreted according to the EUCAST breakpoints as updated in 2016 (http://www.eucast.org). MICs were determined using the Etest technique (bioMérieux).

Carbapenemase detection.Carbapenemase activity was evidenced in Enterobacteriaceae using the updated version of the Carba NP test (12), and carbapenemase genes were detected using an in-house PCR-sequencing approach directly on colonies (6, 15, 21).

Automated DNA extraction from cultured bacterial colonies.Fresh overnight bacterial cultures (on MH agar) were used for DNA extraction with a QIAamp DNA minikit (Qiagen, Les Ulis, France) and the QIAcube instrument (Qiagen, Courtaboeuf, France). A full loop of colonies was suspended in 180 μl of tissue lysis (ATL; Qiagen) buffer and processed in the automated extractor according to the protocol suggested by the manufacturer for bacterial pellets. Total extraction time was 1 h. Five microliters of this DNA was used as sample in a 20-μl PCR mixture.

Whole-cell DNA extracted by boiling.One colony of each bacterial strain was suspended in 100 μl of distilled water. After 10 min at 100°C on a dry heating block (Thermo Fisher), the suspensions were centrifuged (1 min at 10,000 rpm). Total extraction time was 11 min. Five microliters of supernatant was used in a 20-μl PCR assay mixture.

Direct amplification.One colony was pricked three times with the tip of three loops of 1 μl each, and bacteria of each tip were dispersed directly into each PCR tube containing 20 μl of PCR mix (see Tables 2 and S5).

Real-time PCR assay (Amplidiag CarbaR+VRE assay).The Amplidiag CarbaR+VRE multiplex amplification was performed to detect carbapenemase (and vancomycin resistance) genes using a fluorophore-labeled TaqMan probe (Fig. 1). The markers detected by the assay are listed in Table S6. Amplidiag CarbaR+VRE comprises required test reagents for PCR and analysis software. The sample material is DNA extracted from a bacterial culture. Assays are performed in 3 reactions per sample, according to the fluorophores indicated in Table S6. The Amplidiag CarbaR+VRE kit obtained CE marking for in vitro diagnostic kits (CE-IVD) on 8 December 2015. The recommended DNA extraction method for pure culture samples to be used in conjunction with the Amplidiag CarbaR+VRE assay is the “Generic 2.0.1” protocol of NucliSENS easyMAG (bioMérieux) or the Amplidiag Easy system (provided by Mobidiag). Other extraction methods could be used with thorough validation by the users (such as for the QIAcube instrument). Concerning PCR instrumentation, the kit is validated on the Rotorgene (Qiagen), CFX96 (Bio-Rad), and ABI7500 (Applied Biosystems) systems.

FIG 1
FIG 1

Experimental procedures. (A) Experimental setup as recommended by the manufacturer. As DNA extraction is open, the QIAcube automated DNA extractor was used. (B) Boiling DNA extraction. (C) One colony was picked three times in each 20-μl PCR tube. PCR amplification results have been automatically interpreted using the manufacturer's proprietary software. The total duration of the process is also indicated.

The assay is divided into three multiplex PCRs, each of which amplifies a specific group of target genes: (i) Acinetobacter oxacillinase genes (blaOXA-23, blaOXA-40, blaOXA-58, and blaOXA-51 with upstream ISAba1); (ii) blaOXA-48-like, blaNDM, blaVIM, blaIMP, and blaKPC; and (iii) vanA and vanB. Each mix contained 10 μl of 2× PCR master mix, 5 μl of assay mix, and 5 μl of DNA (or a bacterial suspension in water for direct amplification assay on one colony pricked three times). The real-time PCR Rotorgene 6000 amplification-detection system (Qiagen) was used. The total run time was 85 min, for 45 cycles of amplifications (recommended by the manufacturer), and 60 min when reducing the number of cycles to 30 (for time optimization), and analysis was available immediately after the run. Results were automatically interpreted using the proprietary analyzer software that analyzes the quantitative PCR (qPCR) raw data obtained in each experiment. To prevent cross talk, the users have to calibrate the system with a DNA calibrator as recommended. The results were represented in several ways: a plate, a graph, and a table panel. The plate panel contains positive or negative symbols and possible warnings and errors related to the analyzed well. The graph panel contains normalized fluorescence intensity and cycle numbers. The table panel contains all results (positive, negative, quantification cycle [Cq] values, warnings, and errors). The software considers that the sample is positive when the Cq is lower than 35.

Statistical analysis.The sensitivity, specificity, positive predictive value, and negative predictive value of the Amplidiag CarbaR+VRE assay were calculated with their respective 95% confidence intervals (95% CIs) using the free software vassarStats (website for statistical computation) at http://vassarstats.net/. The gold standard was PCR followed by sequencing.

RESULTS AND DISCUSSION

The Amplidiag CarbaR+VRE has been tested on a collection of 100 characterized isolates expressing various β-lactamases and on 200 consecutive enterobacterial isolates with reduced susceptibility to at least one carbapenem (imipenem, meropenem, or ertapenem) according to EUCAST guidelines as updated in 2016 (http://www.eucast.org) and referred to the Associated French National Reference Center (F-NRC) for Antimicrobial Resistance dedicated to carbapenem-resistant Enterobacteriaceae from 20 January to 10 February 2016. The markers detected by this assay are blaKPC-like, blaNDM-like, blaVIM-like, blaIMP-like, and blaOXA-48-like; Acinetobacter-related OXA genes, including blaOXA-23, blaOXA-24/-40, blaOXA-58, and blaOXA-51 with upstream ISAba1; and vanA and vanB for enterococci. The last parameter was not evaluated here.

Retrospective evaluation of a collection of well-characterized Enterobacteriaceae, P. aeruginosa, and A. baumannii isolates.During this retrospective evaluation, the Amplidiag CarbaR+VRE assay was used as recommended by the manufacturer, on QIAcube-extracted DNA recovered from fresh overnight bacterial colonies.

In Enterobacteriaceae, the Amplidiag CarbaR+VRE assay detected all KPC, NDM, VIM, IMP, and OXA-48 producers. In addition, strains that produce an OXA-48 variant with carbapenemase activity, including OXA-162, -181, -204, -232, and -244, were also accurately detected (see Table S2 in the supplemental material). In multiple carbapenemase producers, all resistance determinants were correctly identified (Table 1). None of the non-carbapenemase producers gave positive PCR results, except one Serratia marcescens isolate that produced OXA-405, an OXA-48 variant that lacked carbapenemase activity but gained efficient expanded-spectrum cephalosporin hydrolytic activity due to a 4-amino-acid deletion in the active site (Table 1) (24). Consequently, and as already observed with several other molecular assays (23), this OXA-405-producing S. marcescens isolate was considered a false positive. Surprisingly, as opposed to most of the other molecular assays (23), OXA-163, another OXA-48 variant with a slightly different 4-amino-acid deletion in the active site but also lacking carbapenem-hydrolytic activity, was not detected and thus, this isolate was correctly identified as a non-carbapenemase producer (Table S2). Using this strain collection (n = 50), the overall sensitivity and specificity were 100% (95% confidence interval [95% CI] = 85.6% to 99.9%) and 88.9% (95% CI = 50.7% to 99.4%) for the detection of carbapenemase-producing Enterobacteriaceae (CPE), respectively (Table 1).

View this table:
TABLE 1

Global performance of the Amplidiag CarbaR+VRE assaya

Concerning Pseudomonadaceae, all KPC, VIM, and IMP producers were correctly detected (Table S3). As claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2 and GES-5), GIM-1, AIM-1, SPM-1, DIM-1, and OXA-198 were not detected (Table S3). All non-carbapenemase producers gave a negative result. Excluding strains producing rare carbapenemases (7/30) that are not targeted by the assay, the overall sensitivity and specificity were 100% (95% CI = 71.7% to 100%) and 100% (95% CI = 65.6% to 100%) for the detection of carbapenemase-producing Pseudomonadaceae, respectively (Table 1).

The F-NRC for multidrug-resistant (MDR) P. aeruginosa isolates showed that between 2013 and 2016 most of the carbapenem resistance was due to OprD mutations (76%) and only 24% was due to carbapenemases. VIM (72.8%), KPC (0.4%), and IMP (15.6%) enzymes would have been identified with this assay, while DIM (0.2%) and GES-carbapenemases (11%) would have been missed (http://www.cnr-resistance-antibiotiques.fr/ressources/pages/Rapport_CNR_2016.pdf). Thus, 88.8% of the currently spreading carbapenemases in P. aeruginosa in France would be identified with the Amplidiag CarbaR+VRE assay.

Among A. baumannii isolates, the Amplidiag CarbaR+VRE assay could detect all OXA-23-like, OXA-24/-40-like, and OXA-58-like enzymes as well as overexpression of the chromosomally encoded OXA-51-like β-lactamases, due to the presence of ISAba1 upstream of the blaOXA-51-like genes (Table S4). As claimed by the manufacturer, carbapenemases of GES type (e.g., GES-14) and of the OXA-143 group (e.g., OXA-143 and OXA-243) were not detected (Table S4). Of note, carbapenem-resistant A. baumannii isolates have become increasingly frequently isolated in Brazil, and those producing OXA-23 and OXA-143-like enzymes are the most prevalent (25). Excluding strains producing a rare carbapenemase (3/20) that is not targeted by the assay, the overall sensitivity and specificity were 100% (95% CI = 74.7% to 100%) and 100% (95% CI = 19.8% to 100%) for the detection of carbapenemase-producing A. baumannii, respectively (Table S4). During 2013 to 2016, the F-NRC for A. baumannii revealed that most of the carbapenem resistance was due to OXA-23 producers (84,4%), followed by producers of OXA-40 (5.4%), OXA-58 (2.2%), NDM (3.6%), and ISAba1-OXA-51 (4.4%) (http://www.cnr-resistance-antibiotiques.fr/ressources/pages/Rapport_CNR_2016.pdf). Thus, using the Amplidiag CarbaR+VRE assay, all the carbapenemase-producing A. baumannii isolates would have been identified.

One of the limitations of the Amplidiag CarbaR+VRE assay is that it gives only a “positive” or “negative” answer for Acinetobacter oxacillinases with carbapenemase activity (AcOXA) without any distinction between the variants OXA-23, OXA-40, OXA-58, and ISAba1-OXA-51. Moreover, if the blaOXA-51 gene is not located downstream of an ISAba1 sequence, then it is not detected. However, infection control measures with imipenem-resistant Acinetobacter (carbapenem-resistant A. baumannii [CRAB]) would be the same whatever the resistance mechanism involved (26).

Prospective evaluation of enterobacterial isolates with reduced susceptibility to at least one carbapenem.From 20 January to 10 February 2016, 200 consecutive isolates with reduced susceptibility to at least one carbapenem (imipenem, meropenem, and/or ertapenem) were referred to the F-NRC for Antibiotic Resistance for evaluation. Reidentification of these 200 isolates revealed 198 Enterobacteriaceae and 2 A. baumannii isolates. They corresponded to 101 non-CP-GNB and 99 CP-GNB as revealed by the Carba NP test and subsequent in-house PCR analysis. The Amplidiag CarbaR+VRE assay performed on QIAcube-extracted whole DNA could detect all NDM (n = 12), VIM (n = 2), IMP (n = 1), OXA-48 (n = 83), and NDM plus OXA-48 (n = 1) isolates, including several variants of these enzymes (NDM-1, NDM-4, IMP-10, VIM-1, VIM-19, OXA-181, OXA-204, and OXA-244). In addition, both blaOXA-48 and blaNDM genes were correctly detected by the Amplidiag CarbaR+VRE assay in one Klebsiella pneumoniae isolate that coexpressed the two enzymes. Among the non-carbapenemase producers, 8 gave positive PCR results for OXA-48, VIM, and/or NDM with threshold cycle (CT) values ranging from 35 to 40. These isolates were considered contaminations during DNA extraction. Indeed, CTs of true-positive isolates were below 20. Repeated QIAcube DNA extraction and PCRs directly done on colonies of these 8 isolates grown on MH medium gave a PCR-negative result. K. pneumoniae was the most prevalent species (n = 73), including 53.4% that were carbapenemase producers (29 OXA-48, 9 NDM, and 1 VIM-19). Among Escherichia coli isolates, 67.5% (27/40) produced a carbapenemase. The percentage of carbapenemase producers was lower in Enterobacter spp., with only 21.8% (12/55) of the isolates, which is in line with the French epidemiology (6). The other isolates were 7 Klebsiella oxytoca (5 producing OXA-48), 1 VIM-1-producing Raoultella ornithinolytica, 2 Morganella morganii (1 producing NDM-1), 4 Serratia marcescens (1 producing OXA-48 and 1 producing IMP-10), and 14 Citrobacter (12 producing OXA-48) isolates. Noticeably, the 2 Hafnia alvei and the 2 A. baumannii isolates were non-CP-GNB. The performances of the Amplidiag CarbaR+VRE assay in the prospective study of 200 isolates referred to the F-NRC were 100% (95% CI = 95.4% to 100%) for sensitivity, 100% (95% CI = 95.3% to 100%) for specificity, 100% (95% CI = 95.4% to 100%) for positive predictive value, and 100% (95% CI = 95.3% to 100%) for negative predictive value, respectively (Table 1).

When these results were extrapolated to the global French epidemiology (6) the Amplidiag CarbaR+VRE assay would have been able to identify 99.57% (3,306/3,320) of the CPE, missing only 13 IMI producers and one FRI-1 producer (27) (Table 1) and falsely detecting the unique OXA-405-producing S. marcescens as a carbapenemase producer (24).

Detection of OXA carbapenemases in P. mirabilis.The OXA-23 and OXA-58 carbapenemases, which are highly prevalent in carbapenem-resistant Acinetobacter spp., are still considered to be extremely rare in Enterobacteriaceae. Consequently, the molecular tests that are commercially available for the detection of CPE usually do not include blaOXA-23 and blaOXA-58 genes in their targeted genes (19, 20). However, OXA-23- and OXA-58-producing P. mirabilis isolates have been recently described not only in Europe (28–31) but also in other enterobacterial isolates from different countries and continents: Turkey (32), Africa (33), and India (34). It has been suggested (30) that their prevalence might be underestimated due to detection difficulties related to (i) the very weak hydrolysis activity of these carbapenemases and (ii) the intrinsic decreased susceptibility to imipenem (but not ertapenem) of P. mirabilis. To assess whether the Amplidiag CarbaR+VRE assay might detect these strains, we have tested 10 OXA-producing (8 OXA-23 and 2 OXA-58) P. mirabilis isolates and two negative controls (Table S1). All OXA producers were correctly identified. The Amplidiag CarbaR+VRE assay is the first assay able to detect the five most common carbapenemases and the OXA carbapenemases preferentially found in A. baumannii. The use of this assay would allow identification of these “Acinetobacter-related” enzymes not only in P. mirabilis but also in other enterobacterial isolates (32–34), allowing evaluation of the current spread of these resistance determinants and assessment of the risk of further dissemination of these enzymes in Enterobacteriaceae.

Performance of the Amplidiag CarbaR+VRE assay directly on bacterial colonies.The Amplidiag CarbaR+VRE assay allowed detection of the presence of a carbapenemase within a 3-hour period, using the recommended protocol of the manufacturer (Fig. 1). To see whether this time could be reduced, the Amplidiag CarbaR+VRE PCR was directly performed on colonies grown on Muller-Hinton (MH) medium (representing bacteria from a suspicious antibiogram) and on ChromID Carba Smart medium (a medium that is now classically used to screen for CPE).

For bacterial colonies grown on MH medium and directly suspended in the Amplidiag CarbaR+VRE PCR mix, correct results were obtained for all tested strains (Table S5) compared to the manufacturer-recommended protocol (i.e., DNA extraction using the QIAamp DNA extraction kit), with CT values below 20. Thus, in addition to the avoidance of DNA extraction steps, this protocol optimization also allowed the reduction of the total number of cycles to 30 (instead of 45 in the original protocol), leading to a time-to-result of less than 1 h 10 min instead of 3 h (Fig. 1).

When applied on bacterial colonies grown on ChromID Carba Smart medium, the Amplidiag CarbaR+VRE yielded variable results (Table 2). Although P. aeruginosa isolates gave correct results, several false-positive results were obtained with Enterobacteriaceae (Table 2). Assuming that the pigmentation of enterobacterial isolates on ChromID Carba Smart medium might interfere with direct PCR, a fast (11-min) and cheap whole-DNA boiling extraction was performed. By directly applying 5 μl of this boiling extracted DNA to the Amplidiag CarbaR+VRE PCR mix, correct results were then obtained for all tested Enterobacteriaceae (Table 2) Thus, the time-to-result was slightly increased to 1 h 11 min, which is still compatible with a short time-to-result. In addition, this low-cost DNA extraction technique leads to a drastic decrease in the global cost of the test. However, the presence of vancomycin (Van) primers in the present PCR mix is not appropriate for a study on colonies, because carbapenemase genes will be searched for in Gram-negative bacteria and vancomycin resistance genes will be searched for in Gram-positive bacteria. This PCR mix has been developed for an application to complex clinical samples such as rectal swabs or blood cultures.

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TABLE 2

Results of the Amplidiag CarbaR+VRE assay on bacterial colonies recovered from ChromID Carba Smart medium directly from colonies versus DNA extraction (QIAamp DNA extraction or boiling extract)

Conclusion.Our study demonstrated that the Amplidiag CarbaR+VRE assay is well adapted to the French epidemiology of CP-GNB, which reflects the epidemiology of many European countries. Overall, and when excluding the nontargeted less-prevalent carbapenemases in CP-GNB (10/100), the Amplidiag CarbaR+VRE performances reached 100% (95% CI = 91.1% to 99.9%) and 99% (95% CI = 74.1% to 99.8%) sensitivity and specificity, respectively. The main advantage of this test is that it contains a large panel of targeted resistance determinants. Indeed, it detects the main carbapenemases encountered in Enterobacteriaceae (KPC, NDM, VIM, IMP, and OXA-48-like), but it also identifies the most widespread OXA carbapenemases usually identified in A. baumannii (OXA-23-like, OXA-24/-40-like, and OXA-58-like). This last panel might be increasingly important in the future with the recent reports of OXA-23- and OXA-58-producing P. mirabilis and E. coli (28–33).

As for most molecular assays, mutation and/or polymorphisms in the primer/probe binding region of the targeted gene may lead to false-negative results, and thus, new emerging variants may not be detected. Regular testing of novel variants is required to assess sensitivity and specificity of this assay in a constantly evolving carbapenemase field. The main drawback of this assay compared to other molecular techniques (23) is that it requires pure cultures and DNA extraction steps before analysis. The DNA extraction step significantly increases the global cost of the test since ready-to-use kits are recommended by the manufacturer. However, we demonstrated that a rapid and very cheap DNA extraction by boiling might be accurate enough to recover DNA extract and obtain adequate results. In addition, we also showed that the Amplidiag CarbaR+VRE assay might be directly performed on bacterial colonies grown on MH agar, avoiding any additional DNA extraction step. Of note, our results suggested that at least the boiling DNA extraction step is crucial for bacterial colonies recovered from chromogenic media. Other limitations of the current test are that it is not validated directly on stool samples and that it lacks random access. Thus, the use of the Amplidiag CarbaR+VRE assay for the investigation of an outbreak will require at least 24 or 48 h for the culture, and the global cost of this assay will be approximately 15 euros, but the assay might efficiently confirm the presence of carbapenemase production and identify its determinant if targeted.

ACKNOWLEDGMENTS

We are thankful to Elodie Creton and Garance Cotellon from the French National Reference Center for Antibiotic Resistance.

This work was supported by the Assistance Publique-Hôpitaux de Paris (AP-HP), the University Paris-Sud, and the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT) supported by a grant from the French National Research Agency (ANR-10-LABX-33) and by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) DesInMBL (ANR-14-JAMR-002).

L.D. is coinventor of the Carba NP test, the patent for which has been licensed to bioMérieux (La Balmes les Grottes, France).

FOOTNOTES

    • Received 25 July 2017.
    • Returned for modification 20 August 2017.
    • Accepted 2 January 2018.
    • Accepted manuscript posted online 5 January 2018.

  • Supplemental material for this article may be found at https://doi.org/10.1128/JCM.01092-17.

REFERENCES

  1. 1.↵
    1. Logan LK,
    2. Weinstein RA
    . 2017. The epidemiology of carbapenem-resistant Enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis 215:S28–S36. doi:10.1093/infdis/jiw282.
    OpenUrlCrossRef
  2. 2.↵
    1. Gniadek TJ,
    2. Carroll KC,
    3. Simner PJ
    . 2016. Carbapenem-resistant non-glucose-fermenting Gram-negative bacilli: the missing piece to the puzzle. J Clin Microbiol 54:1700–1710. doi:10.1128/JCM.03264-15.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Queenan AM,
    2. Bush K
    . 2007. Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev 20:440–458. doi:10.1128/CMR.00001-07.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Albiger B,
    2. Glasner C,
    3. Struelens MJ,
    4. Grundmann H,
    5. Monnet DL
    , European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) Working Group. 2015. Carbapenemase-producing Enterobacteriaceae in Europe: assessment by national experts from 38 countries. Euro Surveill 20(45). doi:10.2807/1560-7917.ES.2015.20.45.30062.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Potron A,
    2. Poirel L,
    3. Nordmann P
    . 2015. Emerging broad-spectrum resistance in Pseudomonas aeruginosa and Acinetobacter baumannii: mechanisms and epidemiology. Int J Antimicrob Agents 45:568–585. doi:10.1016/j.ijantimicag.2015.03.001.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Bonnin RA,
    2. Nordmann P,
    3. Poirel L
    . 2013. Screening and deciphering antibiotic resistance in Acinetobacter baumannii: a state of the art. Expert Rev Anti Infect Ther 11:571–583. doi:10.1586/eri.13.38.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Dortet L,
    2. Cuzon G,
    3. Ponties V,
    4. Nordmann P
    . 2017. Trends in carbapenemase-producing Enterobacteriaceae, France, 2012 to 2014. Euro Surveill 22(6):30461. doi:10.2807/1560-7917.ES.2017.22.6.30461.
    OpenUrlCrossRef
  8. 8.↵
    1. Lutgring JD,
    2. Limbago BM
    . 2016. The problem of carbapenemase-producing-carbapenem-resistant-Enterobacteriaceae detection. J Clin Microbiol 54:529–534. doi:10.1128/JCM.02771-15.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Maurer FP,
    2. Castelberg C,
    3. Quiblier C,
    4. Bloemberg GV,
    5. Hombach M
    . 2015. Evaluation of carbapenemase screening and confirmation tests with Enterobacteriaceae and development of a practical diagnostic algorithm. J Clin Microbiol 53:95–104. doi:10.1128/JCM.01692-14.
    OpenUrlCrossRef
  10. 10.↵
    1. Dortet L,
    2. Cuzon G,
    3. Plesiat P,
    4. Naas T
    . 2016. Prospective evaluation of an algorithm for the phenotypic screening of carbapenemase-producing Enterobacteriaceae. J Antimicrob Chemother 71:135–140. doi:10.1093/jac/dkv308.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Dortet L,
    2. Bernabeu S,
    3. Gonzalez C,
    4. Naas T
    . 2017. Comparison of two phenotypic algorithms to detect carbapenemase-producing Enterobacteriaceae. Antimicrob Agents Chemother 61:e00796-17. doi:10.1128/AAC.00796-17.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Dortet L,
    2. Agathine A,
    3. Naas T,
    4. Cuzon G,
    5. Poirel L,
    6. Nordmann P
    . 2015. Evaluation of the RAPIDEC CARBA NP, the Rapid CARB Screen and the Carba NP test for biochemical detection of carbapenemase-producing Enterobacteriaceae. J Antimicrob Chemother 70:3014–3022. doi:10.1093/jac/dkv213.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Lasserre C,
    2. De Saint Martin L,
    3. Cuzon G,
    4. Bogaerts P,
    5. Lamar E,
    6. Glupczynski Y,
    7. Naas T,
    8. Tande D
    . 2015. Efficient detection of carbapenemase activity in Enterobacteriaceae by matrix-assisted laser desorption ionization-time of flight mass spectrometry in less than 30 minutes. J Clin Microbiol 53:2163–2171. doi:10.1128/JCM.03467-14.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Bogaerts P,
    2. Yunus S,
    3. Massart M,
    4. Huang TD,
    5. Glupczynski Y
    . 2016. Evaluation of the BYG Carba test, a new electrochemical assay for rapid laboratory detection of carbapenemase-producing Enterobacteriaceae. J Clin Microbiol 54:349–358. doi:10.1128/JCM.02404-15.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Gauthier L,
    2. Bonnin RA,
    3. Dortet L,
    4. Naas T
    . 2017. Retrospective and prospective evaluation of the carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae. PLoS One 12:e0170769. doi:10.1371/journal.pone.0170769.
    OpenUrlCrossRef
  16. 16.↵
    1. Bernabeu S,
    2. Dortet L,
    3. Naas T
    . 2017. Evaluation of the β-CARBA test, a colorimetric test for the rapid detection of carbapenemase activity in Gram-negative bacilli. J Antimicrob Chemother 72:1646–1658. doi:10.1093/jac/dkx061.
    OpenUrlCrossRef
  17. 17.↵
    1. Boutal H,
    2. Naas T,
    3. Devilliers K,
    4. Oueslati S,
    5. Dortet L,
    6. Bernabeu S,
    7. Simon S,
    8. Volland H
    . 2017. Development and validation of a lateral flow immunoassay for the rapid detection of NDM-producing Enterobacteriaceae. J Clin Microbiol 55:2018–2029. doi:10.1128/JCM.00248-17.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Glupczynski Y,
    2. Jousset A,
    3. Evrard S,
    4. Bonnin RA,
    5. Huang TD,
    6. Dortet L,
    7. Bogaerts P,
    8. Naas T
    . 2017. Prospective evaluation of the OKN K-SeT assay, a new multiplex immunochromatographic test for the rapid detection of OXA-48-like, KPC and NDM carbapenemases. J Antimicrob Chemother 72:1955–1960. doi:10.1093/jac/dkx089.
    OpenUrlCrossRef
  19. 19.↵
    1. Findlay J,
    2. Hopkins KL,
    3. Meunier D,
    4. Woodford N
    . 2015. Evaluation of three commercial assays for rapid detection of genes encoding clinically relevant carbapenemases in cultured bacteria. J Antimicrob Chemother 70:1338–1342. doi:10.1093/jac/dku571.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Hoyos-Mallecot Y,
    2. Ouzani S,
    3. Dortet L,
    4. Fortineau N,
    5. Naas T
    . 2017. Performance of the Xpert Carba-R v2 in the daily workflow of a hygiene unit in a country with a low prevalence of carbapenemase-producing Enterobacteriaceae. Int J Antimicrob Agents 49:774–777. doi:10.1016/j.ijantimicag.2017.01.025.
    OpenUrlCrossRef
  21. 21.↵
    1. Naas T,
    2. Cuzon G,
    3. Bogaerts P,
    4. Glupczynski Y,
    5. Nordmann P
    . 2011. Evaluation of a DNA microarray (Check-MDR CT102) for rapid detection of TEM, SHV, and CTX-M extended-spectrum β-lactamases and of KPC, OXA-48, VIM, IMP, and NDM-1 carbapenemases. J Clin Microbiol 49:1608–1613. doi:10.1128/JCM.02607-10.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Ellington MJ,
    2. Ekelund O,
    3. Aarestrup FM,
    4. Canton R,
    5. Doumith M,
    6. Giske C,
    7. Grundman H,
    8. Hasman H,
    9. Holden MT,
    10. Hopkins KL,
    11. Iredell J,
    12. Kahlmeter G,
    13. Köser CU,
    14. MacGowan A,
    15. Mevius D,
    16. Mulvey M,
    17. Naas T,
    18. Peto T,
    19. Rolain JM,
    20. Samuelsen Ø,
    21. Woodford N
    . 2017. The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee. Clin Microbiol Infect 23:2–22. doi:10.1016/j.cmi.2016.11.012.
    OpenUrlCrossRef
  23. 23.↵
    1. Dortet L,
    2. Fusaro M,
    3. Naas T
    . 2016. Improvement of the Xpert Carba-R kit for the detection of carbapenemase-producing Enterobacteriaceae. Antimicrob Agents Chemother 60:3832–3837. doi:10.1128/AAC.00517-16.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Dortet L,
    2. Oueslati S,
    3. Jeannot K,
    4. Tandé D,
    5. Naas T,
    6. Nordmann P
    . 2015. Genetic and biochemical characterization of OXA-405, an OXA-48-type extended-spectrum β-lactamase without significant carbapenemase activity. Antimicrob Agents Chemother 59:3823–3828. doi:10.1128/AAC.05058-14.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Mostachio AK,
    2. Levin AS,
    3. Rizek C,
    4. Rossi F,
    5. Zerbini J,
    6. Costa SF
    . 2012. High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil. Int J Antimicrob Agents 39:396–401. doi:10.1016/j.ijantimicag.2012.01.021.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Munoz-Price LS,
    2. Weinstein RA
    . 2008. Acinetobacter infection. N Engl J Med 358:1271–1281. doi:10.1056/NEJMra070741.
    OpenUrlCrossRefPubMedWeb of Science
  27. 27.↵
    1. Naas T,
    2. Dortet L,
    3. Iorga BI
    . 2016. Structural and functional aspects of class A carbapenemases. Curr Drug Targets 17:1006–1028. doi:10.2174/1389450117666160310144501.
    OpenUrlCrossRefPubMed
  28. 28.↵
    1. Bonnet R,
    2. Marchandin H,
    3. Chanal C,
    4. Sirot D,
    5. Labia R,
    6. De Champs C,
    7. Jumas-Bilak E,
    8. Sirot J
    . 2002. Chromosome-encoded class D beta-lactamase OXA-23 in Proteus mirabilis. Antimicrob Agents Chemother 46:2004–2006. doi:10.1128/AAC.46.6.2004-2006.2002.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Österblad M,
    2. Karah N,
    3. Halkilahti J,
    4. Sarkkinen H,
    5. Uhlin BE,
    6. Jalava J
    . 2016. Rare detection of the Acinetobacter class D carbapenemase blaOXA-23 gene in Proteus mirabilis. Antimicrob Agents Chemother 60:3243–3245. doi:10.1128/AAC.03119-15.
    OpenUrlFREE Full Text
  30. 30.↵
    1. Girlich D,
    2. Bonnin RA,
    3. Bogaerts P,
    4. De Laveleye M,
    5. Huang DT,
    6. Dortet L,
    7. Glaser P,
    8. Glupczynski Y,
    9. Naas T
    . 2017. Chromosomal amplification of the blaOXA-58 carbapenemase gene in a Proteus mirabilis clinical isolate. Antimicrob Agents Chemother 61:e01697-16. doi:10.1128/AAC.01697-16.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Lange F,
    2. Pfennigwerth N,
    3. Gerigk S,
    4. Gohlke F,
    5. Oberdorfer K,
    6. Purr I,
    7. Wohanka N,
    8. Roggenkamp A,
    9. Gatermann SG,
    10. Kaase M
    . 2017. Dissemination of blaOXA-58 in Proteus mirabilis isolates from Germany. J Antimicrob Chemother 72:1334–1339. doi:10.1093/jac/dkw566.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Budak S,
    2. Aktas Z,
    3. Oncul O,
    4. Acar A,
    5. Ozyurt M,
    6. Turhan V,
    7. Gorenek L
    . 2012. Detection of Acinetobacter baumannii derived OXA-51 and OXA-58 carbapenemase genes in enteric bacteria: a new dimension on carbapenemase resistance. Clin Microbiol Infect 18(Suppl):311.
    OpenUrlCrossRefPubMed
  33. 33.↵
    1. Leski TA,
    2. Bangura U,
    3. Jimmy DH,
    4. Ansumana R,
    5. Lizewski SE,
    6. Li RW,
    7. Stenger DA,
    8. Taitt CR,
    9. Vora GJ
    . 2013. Identification of blaOXA-51-like, blaOXA-58, blaDIM-1, and blaVIM carbapenemase genes in hospital Enterobacteriaceae isolates from Sierra Leone. J Clin Microbiol 51:2435–2438. doi:10.1128/JCM.00832-13.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Paul D,
    2. Ingti B,
    3. Bhattacharjee D,
    4. Maurya AP,
    5. Dhar D,
    6. Chakravarty A,
    7. Bhattacharjee A
    . 2017. An unusual occurrence of plasmid-mediated blaOXA-23 carbapenemase in clinical isolates of Escherichia coli from India. Int J Antimicrob Agents 49:642–645. doi:10.1016/j.ijantimicag.2017.01.012.
    OpenUrlCrossRef
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KEYWORDS

multiplex PCR
real-time detection
screening
confirmation
carbapenemase
molecular methods

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