The novel, real-time PCR-based GenePOC Carba assay on the microfluidic revogene platform (GenePOC, Québec, Canada; now Meridian Bioscience, Cincinnati, OH, USA) was recently designed for the detection of blaKPC, blaNDM, blaVIM, blaOXA-48-like, and blaIMP.
KEYWORDS: GeneXpert, IMP, KPC, MDR, MRE, NDM, OXA-48, VIM, carbapenemase, multidrug resistant
ABSTRACT
The novel, real-time PCR-based GenePOC Carba assay on the microfluidic revogene platform (GenePOC, Québec, Canada; now Meridian Bioscience, Cincinnati, OH, USA) was recently designed for the detection of blaKPC, blaNDM, blaVIM, blaOXA-48-like, and blaIMP. The goals of this study were to evaluate the performance of this assay, to assess its suitability for the routine microbiology laboratory, and to compare it to the Xpert Carba-R assay for the detection of carbapenemase-producing Enterobacterales (CPE) strains. The Xpert Carba-R assay (Cepheid) and the GenePOC Carba assay were challenged with a collection of 176 clinical Enterobacterales isolates. The collection included 133 CPE strains producing a total of 139 carbapenemases, including VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), and IMP (n = 9). Six isolates produced two different carbapenemases, and 43 carbapenemase-negative isolates were included as negative controls. The overall sensitivity for carbapenemase detection was 96.4% (95% confidence interval [CI], 91.9% to 98.5%) for the Xpert Carba-R assay and 100% (95% CI, 97.3% to 100%) for the GenePOC assay. The four most common carbapenemases (NDM, KPC, OXA-48-like, and VIM) were detected with a sensitivity of 100% (95% CI, 97.1% to 100%) by the two tests, with all double carbapenemase producers being correctly detected by both assays. The sensitivity of the Xpert Carba-R assay for IMP was 44.4% (95% CI, 18.9% to 73.3%), while that of the GenePOC assay was 100% (95% CI, 70.1% to 100%). The specificity of both assays was 100% (95% CI, 91.8% to 100%). The GenePOC Carba assay showed excellent sensitivity and specificity for the five most common carbapenemases, including IMP variants. Its simplicity and short turnaround time make it suitable for use in the routine microbiology laboratory for CPE detection.
INTRODUCTION
The worldwide rise and dissemination of antimicrobial resistance currently constitute a global public health concern, leading to increasingly limited therapeutic options and severely compromising patient outcomes (1). In the β-lactam antibiotic armamentarium, carbapenems represent the last-resort therapy for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. Resistance to carbapenems in Enterobacterales is caused by the hyperproduction of AmpC or extended-spectrum β-lactamases (ESBLs) combined with porin loss or by the expression of carbapenem-hydrolyzing enzymes called carbapenemases (i.e., carbapenemase-producing Enterobacterales [CPE]). Therefore, rapid and accurate identification of carbapenemases is of upmost importance, not only for opposition to CPE spread but also for epidemiological and outbreak-control purposes and clinical decision-making (2). Since most isolates producing OXA-48-like carbapenemase or Klebsiella pneumoniae carbapenemase (KPC) are susceptible to the new cephalosporin-inhibitor combination ceftazidime-avibactam, rapid identification of carbapenemase types can also help to rapidly optimize antibiotic therapy.
A number of phenotypic methods for carbapenemase detection (e.g., modified Hodge test or various combination disk tests) show long turnaround times and variable sensitivity according to carbapenemase type and microorganism. Colorimetric tests, such as the Rapidec Carba NP assay (bioMérieux, Nürtingen, Germany) or the β-Carba assay (Bio-Rad, Marnes-la-Coquette, France), have demonstrated variable sensitivity depending on β-lactamase activity and culture medium used (3, 4). Hydrolysis of carbapenems can also be detected by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (5), but this method is only rarely used in routine microbiology. A recently published carbapenemase inactivation method supplemented with ZnSO4, called zCIM (4), has demonstrated a sensitivity of 97.4% (95% CI, 93.4% to 99.0%) for carbapenemase detection. Some phenotypic tests require an extra overnight culture, however, and most are unable to identify the underlying carbapenemase. Novel immunochromatographic tests (ICTs), such as the Resist-4 O.K.N.V. assay (Coris BioConcept, Gembloux, Belgium) and the Carba-5 assay (NG Biotech, Guipry, France), have been shown to be rapid and cost-effective methods with high sensitivity and specificity (4, 6–8). However, PCR-based assays, although able to detect only targeted genes, currently remain the gold standard for carbapenemase detection. The GenePOC Carba assay (GenePOC, Québec, Canada), now the revogene Carba C assay (Meridian Bioscience, Cincinnati, OH, USA), is a recently developed real-time PCR assay that runs on the centripetal microfluidic revogene platform. It has been designed for the detection of blaKPC, blaNDM, blaVIM, blaOXA-48-like, and blaIMP genes from pure colonies of Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa (9). The aims of this study were to evaluate the performance of this assay, to assess its suitability in the routine microbiology laboratory, and to compare it to the Xpert Carba-R assay for the detection of CPE.
MATERIALS AND METHODS
Bacterial isolates.
A total of 176 clinical Enterobacterales isolates that had been previously characterized by PCR and sequencing of carbapenemase genes were tested with the GenePOC Carba assay. In addition, the results of the GenePOC Carba assay were compared to those of the GeneXpert assay, which had been previously assessed with the same isolate collection (4). All clinical strains were isolated from hospitalized patients in Germany (4, 8, 10–13). The strain collection included 133 CPE isolates producing a total of 139 carbapenemases, including isolates belonging to Ambler classes A (n = 11), B (n = 80), and D (n = 36); 6 isolates produced two different carbapenemases (Table 1; also see Table S1 in the supplemental material), and 43 isolates were carbapenemase negative (Table S2).
TABLE 1.
Overview of the bacterial isolate collection used for the evaluation of the two carbapenemase detection methods
| Isolate characteristic | No. of isolates |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| K. pneumoniae | E. coli | E. cloacae | Citrobacter freundii | Klebsiella aerogenes | Serratia marcescens | Proteus mirabilis | Othera | Subtotal | |
| Carbapenemase positive | 47 | 32 | 23 | 18 | 2 | 5 | 1 | 5 | 133 |
| Ambler class A | 9 | 1 | 1 | 11 | |||||
| KPC | 9 | 1 | 1 | 11 | |||||
| KPC-2 | 8 | 1 | 9 | ||||||
| KPC-3 | 1 | 1 | 2 | ||||||
| Ambler class B | 18 | 13 | 23 | 16 | 5 | 1 | 4 | 80 | |
| IMP | 3 | 1 | 2 | 3 | 9 | ||||
| IMP-1 | 1 | 1 | 2 | ||||||
| IMP-4 | 1 | 1 | |||||||
| IMP-8 | 1 | 1 | |||||||
| IMP-13 | 1 | 1 | |||||||
| IMP-14 | 1 | 1 | |||||||
| IMP-22 | 1 | 1 | |||||||
| IMP-28 | 1 | 1 | |||||||
| IMP-50 | 1 | 1 | |||||||
| NDM | 10 | 8 | 4 | 1 | 1 | 1 | 1 | 26 | |
| NDM-1 | 7 | 4 | 3 | 1 | 1 | 1 | 17 | ||
| NDM-3 | 1 | 1 | |||||||
| NDM-4 | 1 | 1 | |||||||
| NDM-5 | 1 | 1 | 2 | ||||||
| NDM-7 | 1 | 1 | 2 | ||||||
| NDM-8 | 1 | 1 | |||||||
| NDM-9 | 2 | 2 | |||||||
| VIM | 5 | 4 | 19 | 13 | 1 | 3 | 45 | ||
| VIM-1 | 2 | 4 | 9 | 5 | 1 | 21 | |||
| VIM-2 | 1 | 1 | 1 | 1 | 4 | ||||
| VIM-4 | 2 | 2 | 1 | 5 | |||||
| VIM-5 | 1 | 1 | |||||||
| VIM-26 | 2 | 2 | |||||||
| VIM-27 | 1 | 1 | |||||||
| VIM-31 | 1 | 1 | |||||||
| VIM-39 | 3 | 3 | |||||||
| VIM-46 | 1 | 1 | |||||||
| VIM-51 | 1 | 1 | |||||||
| VIM-52 | 1 | 1 | |||||||
| VIM-54 | 1 | 1 | |||||||
| VIM-56 | 1 | 1 | |||||||
| VIM-58 | 1 | 1 | |||||||
| VIM-59 | 1 | 1 | |||||||
| Ambler class D | 17 | 16 | 1 | 2 | 36 | ||||
| OXA | 17 | 16 | 1 | 2 | 36 | ||||
| OXA-48-like | 17 | 16 | 1 | 2 | 36 | ||||
| OXA-48 | 9 | 9 | 2 | 20 | |||||
| OXA-162 | 2 | 1 | 1 | 4 | |||||
| OXA-181 | 3 | 3 | |||||||
| OXA-204 | 1 | 1 | |||||||
| OXA-232 | 1 | 2 | 3 | ||||||
| OXA-244 | 1 | 1 | 2 | ||||||
| OXA-245 | 2 | 2 | |||||||
| OXA-370 | 1 | 1 | |||||||
| Two carbapenemases | 3 | 3 | 6 | ||||||
| OXA-232 + NDM-1 | 1 | 1 | |||||||
| KPC-2 + VIM-1 | 2 | 2 | |||||||
| OXA-181 + NDM-5 | 1 | 1 | |||||||
| OXA-48 + NDM-1 | 1 | 1 | |||||||
| OXA-48 + VIM-1-like | 1 | 1 | |||||||
| Carbapenemase negative | 10 | 20 | 6 | 1 | 4 | 1 | 1 | 43 | |
| Total | 57 | 52 | 29 | 19 | 6 | 5 | 2 | 6 | 176 |
Other species include Escherichia hermannii (VIM-1 [n = 1]), Klebsiella oxytoca (VIM-2 [n = 1] and VIM-4 [n = 1]), Citrobacter braakii (KPC-2 [n = 1]), Raoultella ornithinolytica (NDM-1 [n = 1]), and carbapenemase-negative Klebsiella oxytoca (n = 1).
MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) was employed for species identification of isolates. The determination of MICs for ertapenem, meropenem, and imipenem was performed using MIC test strips (Liofilchem, Roseto degli Abruzzi, Italy). MICs were interpreted according to EUCAST 8.1 breakpoints (http://www.eucast.org/clinical_breakpoints).
The presence of the β-lactamase (bla) genes encoding OXA-48-like, VIM, IMP, NDM, and KPC enzymes was analyzed for all strains by PCR and subsequent DNA Sanger sequencing, as described previously (4). For phenotypic identification of carbapenemases, a combination disk test (KPC and metallo-β-lactamase [MBL] disk kit; Liofilchem, Roseto degli Abruzzi, Italy) was used as recommended by EUCAST (14), with meropenem and one of the following β-lactamase inhibitors: EDTA (MBL), cloxacillin (AmpC), or phenylboronic acid (class A). Additionally, a temocillin disk was employed to screen for OXA-48-like carbapenemases. Carbapenemase-negative isolates were further tested for ESBL and AmpC production. ESBL production was assessed as recommended by EUCAST (14), using a combination disk test with disks for cefotaxime, ceftazidime, and cefepime with or without clavulanate (Mast Diagnostica, Reinfeld, Germany). AmpC production was tested with a cefoxitin-cloxacillin disk test (Liofilchem). Additionally, carbapenemase-negative ESBL-producing isolates were further characterized by PCR (15) and sequencing (Table S3).
For comparison of the two assays, a suspension equivalent to a 0.5 McFarland turbidity standard was inoculated on Mueller-Hinton agar plates (Oxoid, Wesel, Germany) and incubated overnight at 37°C. All tests were performed according to the manufacturer’s recommendations.
Cepheid Xpert Carba-R assay.
A full 10-μl inoculation loop with a bacterial suspension equivalent to a 0.5 McFarland standard was added to the sample reagent. After homogenization by vortex-mixing for 10 s, 1.7 ml was transferred into the Xpert Carba-R cartridge (version 2.0) and run on the GeneXpert platform (Cepheid).
GenePOC Carba assay.
After homogenization by vortex-mixing for 15 s, 15 μl of a bacterial suspension equivalent to a 0.5 McFarland standard was added to the sample buffer tube. After subsequent vortex-mixing for 15 s, 120 to 200 μl of sample buffer was transferred into the sample loading chamber of the microfluidic cartridge (Fig. 1). Following the manufacturer´s instructions, 8 cartridges were used for every run in the revogene instrument, using mock cartridges when fewer than 8 samples were processed.
FIG 1.
Revogene instrument (left) and GenePOC cartridge (right).
Statistics.
Molecular characterization was used as the reference for the calculation of sensitivity and specificity. The 95% confidence intervals (CIs) and the Youden index were also calculated. The Youden index (sensitivity + specificity − 1) calculates the efficiency of a diagnostic test; values range from −1 to 1, with 1 indicating a perfect test.
RESULTS
Of 176 isolates assessed with the two assays, 133 were CPE isolates, producing a total of 139 carbapenemases (Table 1; also see Table S1 in the supplemental material). Among the 43 carbapenemase-negative isolates, 21 (48.8%) were resistant to at least ertapenem, mostly through production of ESBL (n = 7) and/or AmpC (n = 15) in combination with porin loss (Table S2).
The overall sensitivity of the two tests for carbapenemase detection was 100% (95% CI, 97.3% to 100%) for the GenePOC Carba assay and 96.4% (95% CI, 91.9% to 98.5%) for the Xpert Carba-R assay (Table 2). When only the four most common carbapenemases (NDM, KPC, OXA-48-like, and VIM) were considered (n = 130), the sensitivity was 100% (95% CI, 97.1% to 100%) for both tests. All double carbapenemase producers (n = 6) were correctly detected by both assays. While the sensitivity of the Xpert Carba-R assay for IMP detection was 44.4% (95% CI, 18.9% to 73.3%), with only IMP-1, IMP-4, and IMP-28 variants being detected, that of the GenePOC Carba assay was 100% (95% CI, 70.1% to 100%). The specificity of both assays was 100% (95% CI, 91.8% to 100%). The Youden index values were 0.96 and 1 for the Xpert Carba-R and GenePOC Carba assays, respectively.
TABLE 2.
Performance of the two assays for detection of carbapenemase production
| Carbapenemase type | GenePOC Carba assay |
Xpert Carba-R assay |
||||
|---|---|---|---|---|---|---|
| Sensitivity (95% CI) (%) | Specificity (95% CI) (%) | Youden index | Sensitivity (95% CI) (%) | Specificity (95% CI) (%) | Youden index | |
| All carbapenemases (n = 139) | 100 (97.3–100) | 100 (91.8–100) | 1 | 96.4 (91.9–98.5) | 100 (91.8–100) | 0.96 |
| Ambler class A (n = 13) | 100 (77.2–100) | 100 (77.2–100) | ||||
| KPC (n = 13) | 100 (77.2–100) | 100 (77.2–100) | ||||
| KPC-2 (n = 11) | 100 | 100 | ||||
| KPC-3 (n = 2) | 100 | 100 | ||||
| Ambler class B (n = 86) | 100 (95.7–100) | 94.2 (87.1–97.5) | ||||
| NDM (n = 29) | 100 (88.3–100) | 100 (88.3–100) | ||||
| NDM-1 (n = 19) | 100 | 100 | ||||
| NDM-3 (n = 1) | 100 | 100 | ||||
| NDM-4 (n = 1) | 100 | 100 | ||||
| NDM-5 (n = 3) | 100 | 100 | ||||
| NDM-7 (n = 2) | 100 | 100 | ||||
| NDM-8 (n = 1) | 100 | 100 | ||||
| NDM-9 (n = 2) | 100 | 100 | ||||
| VIM (n = 48) | 100 (92.6–100) | 100 (92.6–100) | ||||
| VIM-1 (n = 23) | 100 | 100 | ||||
| VIM-2 (n = 4) | 100 | 100 | ||||
| VIM-4 (n = 5) | 100 | 100 | ||||
| VIM-5 (n = 1) | 100 | 100 | ||||
| VIM-26 (n = 2) | 100 | 100 | ||||
| VIM-27 (n = 1) | 100 | 100 | ||||
| VIM-31 (n = 1) | 100 | 100 | ||||
| VIM-39 (n = 3) | 100 | 100 | ||||
| VIM-46 (n = 1) | 100 | 100 | ||||
| VIM-51 (n = 1) | 100 | 100 | ||||
| VIM-52 (n = 1) | 100 | 100 | ||||
| VIM-54 (n = 1) | 100 | 100 | ||||
| VIM-56 (n = 1) | 100 | 100 | ||||
| VIM-58 (n = 1) | 100 | 100 | ||||
| VIM-59 (n = 1) | 100 | 100 | ||||
| VIM-1-like (n = 1) | 100 | 100 | ||||
| IMP (n = 9) | 100 (70.1–100) | 44.4 (18.9–73.3) | ||||
| IMP-1 (n = 2) | 100 | 100 | ||||
| IMP-4 (n = 1) | 100 | 100 | ||||
| IMP-8 (n = 1) | 100 | 0 | ||||
| IMP-13 (n = 1) | 100 | 0 | ||||
| IMP-14 (n = 1) | 100 | 0 | ||||
| IMP-22 (n = 1) | 100 | 0 | ||||
| IMP-28 (n = 1) | 100 | 100 | ||||
| IMP-50 (n = 1) | 100 | 0 | ||||
| Ambler class D (n = 40) | 100 (91.2–100) | 100 (91.2–100) | ||||
| OXA-48-like (n = 40) | 100 (91.2–100) | 100 (91.2–100) | ||||
| OXA-48 (n = 22) | 100 | 100 | ||||
| OXA-162 (n = 4) | 100 | 100 | ||||
| OXA-181 (n = 4) | 100 | 100 | ||||
| OXA-204 (n = 1) | 100 | 100 | ||||
| OXA-232 (n = 4) | 100 | 100 | ||||
| OXA-244 (n = 2) | 100 | 100 | ||||
| OXA-245 (n = 2) | 100 | 100 | ||||
| OXA-370 (n = 1) | 100 | 100 | ||||
Of all tests performed with the GenePOC Carba assay, 2 carbapenemase-positive Escherichia coli strains required retesting. First, an OXA-232-producing strain that initially tested indeterminate due to technical issues with the cartridge was positive for OXA-48-like in repeat testing. Second, an OXA-48-producing strain that initially tested positive for OXA-48-like plus NDM tested negative for NDM in repeat testing. The same strain tested negative for the MBL phenotype in a combined disk diffusion test and was NDM negative by ICT and PCR.
The procedure for sample preparation was easy to perform, taking around 5 to 10 min per isolate, with a run time of approximately 70 min on the revogene instrument.
DISCUSSION
Highly sensitive and specific point-of-care diagnostic methods that are rapid and easy are urgently needed to guide antimicrobial therapy and to contain the further dissemination of CPE. The GenePOC Carba assay has been recently developed as a microfluidic device system for carbapenemase detection in CPE strains. To the best of our knowledge, this is the first study to assess its performance. By using the same isolate collection, the performance of this assay could be compared to that of the Xpert Carba-R assay, a diagnostic test that is widely used in routine microbiology laboratories for the detection and differentiation of blaKPC, blaNDM, blaVIM, blaOXA-48-like, and blaIMP gene sequences (16).
The overall sensitivity for carbapenemase detection was 100% (95% CI, 97.3% to 100%) for the GenePOC Carba assay and 96.4% (95% CI, 91.9% to 98.5%) for the Xpert Carba-R assay, with the four most common carbapenemases and all double carbapenemase producers being correctly detected by both systems. In addition, both assays showed a specificity of 100% (95% CI, 91.8% to 100%). Our results with the Xpert Carba-R assay are in line with previous studies, in which the recently added OXA-181 and OXA-232 gene sequences were also detected (16–18). Interestingly, the sensitivity of the Xpert Carba-R assay for the IMP variants included in this study was 44.4% (95% CI, 18.9% to 73.3%), with results for IMP-8, IMP-13, IMP-14, IMP-22, and IMP-50 variants being negative after repeated testing. This is in line with the manufacturer´s specifications, which state that only IMP-1-related variants can be detected (16–18). The variants not detected by the Xpert Carba-R assay all had nucleotide identity with IMP-1 of less than 93% (see Table S4 in the supplemental material). In contrast, the GenePOC Carba assay yielded a sensitivity of 100% (95% CI, 70.1% to 100%) for the detection of all IMP variants assessed. Our results with the GenePOC Carba assay are in line with the manufacturer´s specifications regarding the carbapenemase variants that can be detected (Table S5). All IMP variants included in the study have been demonstrated either experimentally or in silico to be detectable by the assay; an exception is IMP-50, which was also detected in this study.
This study included a large variety of molecularly characterized clinical CPE isolates (40 different carbapenemase variants and 11 species). However, because IMP carbapenemases are rare in Germany, not all IMP variants could be included in this study. In addition, further studies are needed to evaluate the performance of the GenePOC Carba assay with Acinetobacter baumannii and Pseudomonas aeruginosa and also directly with clinical specimens. An additional limitation might be that species identification was performed using MALDI-TOF MS, which cannot differentiate reliably between some closely related species such as K. pneumoniae and Klebsiella quasipneumoniae or species of the Enterobacter cloacae complex.
In a previous study by our group (4), all isolates producing IMP were identified only using the β-Carba assay or the zCIM test, which indicate carbapenemase activity but cannot identify the corresponding carbapenemase. ICTs are more rapid and affordable than PCR but are not able to identify all IMP variants; for example, the Carba-5 assay (NG Biotech) could not detect IMP-13, IMP-14, IMP-28, or IMP-50 (4). In contrast to PCR, detection of some carbapenemases by ICTs is influenced by the inoculum, antibiotics, and culture medium (19); additionally, double carbapenemase producers are identified less reliably with ICTs (4).
Rare carbapenemases (e.g., GES, IMI, GIM, and OXA-58) are currently not targeted by the two assays evaluated or most other commercially available PCR assays. Therefore, other diagnostic tests, such as the Carba NP test (20) or the zCIM test (4), which have previously proved to be sensitive for rare carbapenemase detection, are necessary to detect all CPE strains.
In comparison with the Xpert Carba-R assay, the GenePOC Carba assay showed a similar turnaround time and a straightforward sample preparation procedure, with the final results being easy to interpret. The price for one test is slightly lower for the GenePOC Carba assay (€39 [approximately $44]) than for the Xpert Carba-R assay (€45 [approximately $50]).
In conclusion, both assays showed excellent sensitivity and specificity for detection of the four most common carbapenemases. The GenePOC Carba assay showed greater sensitivity (100%) for the detection of IMP variants than did the Xpert Carba-R assay (44.4%), which is interesting for regions in which this carbapenemase is prevalent. Given its user friendliness and short time to result, the GenePOC Carba assay is a suitable test for microbiology laboratories for the identification of CPE.
Supplementary Material
ACKNOWLEDGMENTS
We thank Yvonne Stelzer, Tanja Oestreicher, and Anne Brunke for excellent technical assistance.
This study was supported by grants from GenePOC and the DZIF to A.H. The funders had no role in collection and interpretation of data or the decision to submit the work for publication.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/JCM.00597-19.
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