Abstract
Rapid identification of patients who are colonized with carbapenemase-producing organisms (CPO) is included in multiple national guidelines for containment of these organisms. In a multisite study, we evaluated the performance of the Cepheid Xpert Carba-R assay, a qualitative diagnostic test that was designed for the rapid detection and differentiation of the blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP-1 genes from rectal swab specimens. A double rectal swab set was collected from 383 patients admitted at four institutions (2 in the United States, 1 in the United Kingdom, 1 in Spain). One swab was used for reference culture (MacConkey broth containing 1 mg/liter of meropenem and subcultured to a MacConkey agar plate with a 10-μg meropenem disk) and for sequencing of DNA obtained from carbapenem-nonsusceptible isolates for carbapenemase identification. The other swab was used for the Xpert Carba-R assay. In addition to the clinical rectal swabs, 250 contrived specimens (108 well-characterized CPO and 142 negative controls spiked onto negative rectal swabs) were tested. Overall, 149/633 (23.5%) samples were positive by the Xpert Carba-R assay. In 6 samples, multiple targets were detected (4 VIM/OXA-48, 1 IMP-1/NDM, and 1 NDM/KPC). The Xpert Carba-R assay detected 155 targets (26 IMP-1, 30 VIM, 27 NDM, 33 KPC, 39 OXA-48) within a time range of 32 to 48 min. The sensitivity, specificity, and positive and negative predictive values of the Xpert Carba-R assay compared to those of the reference culture and sequencing results were 96.6% (95% confidence interval [CI], 92.2% to 98.9%), 98.6% (95% CI, 97.1% to 99.4%), 95.3%, and 99.0%, respectively. The Cepheid Xpert Carba-R assay is an accurate and rapid test to identify rectal colonization with CPO, which can guide infection control programs to limit the spread of these organisms.
INTRODUCTION
The global spread of carbapenemase-producing organisms (CPO) has been highlighted by international health authorities as a critical public health concern (1–3). Containment of this spread, currently recommended in North American and European infection control guidelines, includes accurate and rapid identification of colonization with CPO (4, 5). Different methods for detection of CPO in rectal swabs have been used, including culture with specific chromogenic media and in-house and commercial molecular tests (6, 7). Culture-based methods are limited by sensitivity and specificity issues depending on the composition of the medium and the targeted carbapenemases (e.g., KPC, VIM, NDM, IMP, or OXA-48 enzymes), the requirement of 24 to 48 h for growth, and the need for confirmation with phenotypic and/or molecular methods. Moreover, as the accuracy of culture methods is highly dependent on medium formulation (type and concentration of antibiotic supplementation), the increasing diversity of carbapenemase enzymes and their variable expression affect the sensitivity of culture methods (8, 9). In addition, bacterial load in rectal or perirectal swabs also impacts the accuracy of culture-based tests. For these reasons, molecular tests have been developed and claim to overcome most of these limitations. These molecular assays comprise multiplex-PCR-based assays (10, 11), isothermal amplification (LAMP)-based assays (12), and microarray-based assays (13, 14). Some of these assays have been evaluated using only a collection of well-characterized CPO but not with clinical samples and/or with a multisite approach.
In a previous study, we evaluated the sensitivity and specificity of a PCR-based method in a cartridge format (Xpert MDRO assay) for detecting carbapenemase genes in rectal and perirectal swabs run on the GeneXpert platform (Cepheid, Sunnyvale, CA). Testing was performed in parallel with a reference culture method (15). This molecular assay included targets for blaKPC, blaNDM, and blaVIM genes but not for blaOXA-48 and blaIMP-1. In the current study, an updated version of the Xpert Carba-R (Cepheid, Sunnyvale, CA) was evaluated, which included all five gene targets. The evaluation was performed in a multisite prospective study for European marketing authorization under the European directive on in vitro diagnostic medical devices (16).
MATERIALS AND METHODS
Study design, prospective, and contrived specimens.
The study was conducted between July 2013 and February 2014 at two institutions in the United States (David Geffen School of Medicine, University of California, Los Angeles, CA and Westchester Medical Center, Valhalla, NY) and two institutions in Europe (Central Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom and Hospital Ramón y Cajal, Madrid, Spain). Specimens included prospectively recovered rectal swabs (n = 383) from patients who provided informed consent to participate in the study or whose routine care included screening for CPO following local infection control policies. The protocol was approved by each institution's Ethics Committee. A double swab set (Venturi Transystem; Copan, CA) and transport medium (liquid Stuart transport swab; Copan) were used to collect and transport rectal swab specimens from eligible subjects. One swab was used for reference culture, susceptibility testing, and sequencing, and another swab was used for the Xpert Carba-R. To diminish the potential bias of sampling differences, the two swabs were gently rolled against one another before starting the Cepheid Xpert Carba-R assay and culture procedure.
Due to the low prevalence of organisms containing some of the carbapenemase genes, the prospectively collected clinical rectal swab specimens were supplemented with an additional 250 contrived specimens. To prepare the contrived specimens, unique, well-characterized carbapenemase-producing (n = 108) and non-carbapenemase-producing isolates (n = 142) were spiked into the negative rectal swab matrix (Table 1). Fifty percent of the specimens were prepared at concentrations near the analytical limit of detection (LOD) of the Xpert Carba-R assay (ranging from 1.1 × 102 to 1.2 × 103 CFU/swab depending on the carbapenemase gene), and the remaining specimens covered clinically relevant analyte concentrations (1×, 3×, and 10× LOD) (17). Isolates were seeded onto mock rectal swabs that were prepared by dipping swabs into an individual stool matrix that tested negative for carbapenemase genes. Nonseeded swabs that were dipped into individual stool matrix were prepared as controls.
TABLE 1.
Xpert Carba-R assay results by target for clinical and contrived specimens
| Xpert Carba-R assay result | Clinical specimens (n = 383) | Contrived specimens (n = 250) | All specimens (n = 633) |
|---|---|---|---|
| Positive (single and/or combined targets) | 42 | 107 | 149 |
| IMP-1 | 0 | 25 | 25 |
| VIM | 2 | 24 | 26 |
| NDM | 2 | 23 | 25 |
| KPC | 13 | 19 | 32 |
| OXA-48 | 20 | 15 | 35 |
| VIM + OXA-48 | 4 | 0 | 4 |
| NDM + KPC | 1 | 0 | 1 |
| IMP-1 + NDM | 0 | 1 | 1 |
| Negative | 341 | 143 | 484 |
Results from the Xpert Carba-R assay for the clinical and contrived specimens were compared to those of the reference culture and sequencing results.
Reference culture, susceptibility testing, and sequencing.
One of the prospectively collected rectal specimen swabs was placed in 10 ml of MacConkey broth (General Laboratory Products, Yorkville, IL, USA) containing a 10-μg meropenem disk (BD, Franklin Lakes, NJ, USA) and was vortexed. After 20 to 24 h of incubation at 35°C, MacConkey broths were sent from the collecting sites to a central reference laboratory (Clinical Microbiology Institute, OR, USA) for further processing. Upon receipt at the central reference laboratory, an aliquot of 100-μl was removed from the MacConkey broth and inoculated onto a MacConkey agar plate after which a 10-μg meropenem disk was placed in the center of the plate. Colonies on the MacConkey agar plate that grew within 27 mm of the meropenem disk after an overnight incubation at 35°C were further tested for the presence of a carbapenemase, as described by Lolans et al. (18) with some modifications. Meropenem was used instead of ertapenem to improve the specificities for Pseudomonas spp. and Acinetobacter spp., which are intrinsically resistant to ertapenem. Colonies were also tested by a standard disk diffusion method, and those classified as carbapenem-nonsusceptible (i.e., intermediate or resistant to ertapenem, imipenem, or meropenem using the Clinical and Laboratory Standards Institute [CLSI] interpretive criteria [19]) were subjected to bidirectional DNA sequence analyses for the identification of the blaKPC, blaNDM, blaOXA-48, blaIMP-1, and blaVIM genes. The bidirectional DNA sequence analyses were performed at an independent laboratory (ACGT Inc., Wheeling, IL, USA) using primers that were different from those used in the Xpert Carba-R assay.
All contrived swab specimens were processed at the central reference laboratory in the same manner as the clinical rectal swab specimens. They were inoculated into MacConkey broth with a meropenem disk and subsequently subcultured to MacConkey agar. Isolated colonies were tested for susceptibility.
A positive reference culture result for both clinical and contrived specimens was defined as the isolation of a carbapenem-nonsusceptible organism that contained a blaKPC, blaNDM, blaOXA-48, blaIMP-1, and/or blaVIM gene confirmed by DNA sequence analysis. A negative reference culture result was defined as either a reference culture that did not yield any carbapenem-nonsusceptible organisms or the isolation of a carbapenem-nonsusceptible organism that did not contain any of the target carbapenemase genes by DNA sequence analysis.
To minimize bias in specimen analyses, the laboratory personnel performing the reference culture and DNA sequencing were not aware of the Xpert Carba-R assay results.
Xpert Carba-R assay.
Xpert Carba-R assay testing was performed using the second swab from the double swab set with the GeneXpert platform. Testing was performed at the laboratories in each of the 4 participating institutions according to the manufacturer's recommendations and as previously described (15) within 24 to 48 h from collection. The assay has a run time of ∼47 min in the instrument. Quality control for the Xpert Carba-R assays consisted of one positive and one negative control. The former was composed of an Escherichia coli isolate containing a plasmid that included DNA fragments of all five target gene sequences, and the latter was the same E. coli isolate with the cloning vector but without the cloned fragments. The controls (product M219) were produced by Maine Molecular Quality Controls, Inc. (http://www.mmqci.com/qc-m219.php). The two controls were run on each day that specimens were tested. Study specimens were not tested until correct results were obtained for the negative and positive controls.
All contrived swab specimens were tested with the Xpert Carba-R assay in the same manner as the clinical rectal swab specimens.
Data analysis, discrepant results, and statistics.
The Xpert Carba-R assay results were compared with reference culture and DNA sequencing results. An Xpert Carba-R-positive result was considered when the Xpert Carba-R assay detected the presence of at least one carbapenemase gene. For each specimen, results for each of the five carbapenemase target genes included in the Xpert Carba-R assay were reported separately, and these were compared to results for the five carbapenemase target genes obtained from reference culture and DNA sequencing.
A discrepant result was defined as a result obtained with the Xpert Carba-R assay that did not correlate with the results of reference culture and DNA sequencing of the same specimen. Discrepant testing was performed for only those specimens that were positive for a target gene by the Xpert Carba-R assay but did not show growth of colonies by culture. DNA was extracted from the MacConkey broth (100 μl) using the Qiagen DNeasy blood and tissue kit using the protocol for Gram-negative bacteria and amplified by PCR using primers corresponding to the targets that were positive by the Xpert Carba-R assay results. If one of the five targets was identified, bidirectional DNA sequence analysis was performed. A positive result was considered a true positive for discrepant resolution by the reference method in the analysis.
Discrepant testing was not performed for specimens that were negative for all target genes by the Xpert Carba-R assay but showed growth of colonies on the MacConkey agar plate and from which one of the five target genes was identified through DNA sequence analysis.
Sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were also calculated for each target carbapenemase gene (20).
Statistical analysis was performed using 95% confidence intervals (CIs) calculated with the Clopper-Pearson/Fisher exact CI (21) using Minitab version 16 (Minitab, State College, PA). Values for the Kappa coefficient, which gives a measure of the percentage of agreement between the Xpert Carba-R assay and the reference method (after resolution of discrepant results) beyond that expected by chance, were also calculated (22).
RESULTS
Xpert Carba-R assay results with prospective rectal and contrived swab specimens.
A total of 633 samples (383 clinical rectal swabs and 250 contrived specimens) were included. Overall, 149/633 (23.5%) samples were positive by the Xpert Carba-R assay, and 484/633 (76.5%) were negative. The Xpert Carba-R assay amplified 155 positive targets (26 blaIMP-1, 30 blaVIM, 27 blaNDM, 33 blaKPC, 39 blaOXA-48). In 6 samples, multiple targets were detected (4 blaVIM-blaOXA-48, 1 blaIMP-1-blaNDM, 1 blaNDM-blaKPC). Xpert Carba-R results stratified by clinical and contrived specimen type are shown in Table 1. Data analyses included discrepant resolution results (see below). All positive results were obtained within 32 to 48 min.
Comparison of the Xpert Carba-R assay with the reference method.
Results from the Xpert Carba-R assay by individual target compared to the reference method (culture plus sequencing) for all specimens are shown in Table 2. There were a total of 633 specimens, each with results for five individual targets for a total of 3,165 results. Specimens that were negative by both the Xpert Carba-R assay and the reference method (3,004 total) were stratified as follows: 606 for IMP-1, 601 for VIM, 606 for NDM, 599 for KPC, and 592 for OXA-48.
TABLE 2.
Results from the Xpert Carba-R assay and the reference method (culture plus sequencing) by individual target for combined clinical and contrived specimens
| Xpert Carba-R assay | Reference method (culture plus sequencing) |
||||||
|---|---|---|---|---|---|---|---|
| IMP-1 | VIM | NDM | KPC | OXA-48 | Negative | Total | |
| IMP-1 | 26 | 0 | 0 | 0 | 0 | 0 | 26 |
| VIM | 0 | 29 | 0 | 0 | 0 | 1 | 30 |
| NDM | 0 | 0 | 26 | 0 | 0 | 1 | 27 |
| KPC | 0 | 0 | 0 | 29 | 0 | 4 | 33 |
| OXA-48 | 0 | 0 | 0 | 0 | 38 | 1 | 39 |
| Negative | 1 | 2 | 0 | 1 | 2 | 3,004 | 3,010 |
| Total | 27 | 31 | 26 | 30 | 40 | 3,011 | 3,165 |
Table 3 shows the overall performance of Xpert Carba-R compared with that of the reference method. The results were defined as positive for the Xpert Carba-R assay if any of the carbapenemase targets were positive, and they were defined as negative for the Xpert Carba-R assay if all of the carbapenemase targets were negative. On the combined set of clinical and contrived specimens, sensitivity, specificity, PPV, and NPV were 96.6% (95% CI, 92.2% to 98.9%), 98.6% (95% CI, 97.1% to 99.4%), 95.3%, and 99.0%, respectively. The Kappa index for this comparison was 0.933 (0.900 to 0.967).
TABLE 3.
Overall Xpert Carba-R performance versus that of the reference method (culture plus sequencing) for combined clinical and contrived specimens
| Xpert Carba-R assay | Reference method (culture plus sequencing) |
||
|---|---|---|---|
| No. positive | No. negative | Total No. | |
| Positive | 142 | 7 | 149 |
| Negative | 6 | 478 | 484 |
| Total | 148 | 485 | 633 |
The performance of the Xpert Carba-R assay for different targets, including CI, is shown in Table 4. Results included in Tables 2 to 4 were also analyzed when discrepant results were resolved (see below).
TABLE 4.
Summary of Xpert Carba-R performance for different carbapenemase targets versus that of the reference method (culture plus sequencing) for combined clinical and contrived specimens
| Target gene | Sensitivity (% [95% CI]) | Specificity (% [95% CI]) | PPV (%) | NPV (%) |
|---|---|---|---|---|
| IMP-1 | 96.3 (81.0–99.9) | 100 (99.4–100) | 100 | 99.8 |
| VIM | 93.5 (78.6–99.2) | 99.8 (99.1–100) | 96.7 | 99.7 |
| NDM | 100 (86.8–100) | 99.8 (99.1–100) | 96.3 | 100 |
| KPC | 96.7 (82.8–99.9) | 99.3 (98.3–99.8) | 87.9 | 99.8 |
| OXA-48 | 95.0 (83.1–99.4) | 99.8 (99.1–100) | 97.4 | 99.7 |
Discrepant results and resolution.
Six specimens (4 prospective and 2 contrived specimens) were negative for all target genes by Xpert Carba-R assay but were positive by the reference method (culture and DNA sequencing) and were considered to be false negatives. Bidirectional DNA sequence analysis identified 1 blaIMP-1, 2 blaVIM, 1 blaKPC, and 2 blaOXA-48 genes (Table 2). In addition, 18 specimens (15 prospective and 3 contrived specimens) were positive for at least one target gene by the Xpert Carba-R assay and were negative by the reference method (Table 5). Eleven of the 18 specimens were considered to be true positives after discrepant analyses, which included direct PCR analysis and sequencing from the MacConkey broth. Of these 11 specimens, there were 5 out of the 6 specimens in which multiple carbapenemase targets were detected by Xpert Carba-R assay. The remaining 7 specimens were considered to be false positives, as no PCR amplification was obtained from testing the MacConkey broth (Table 5). Targets detected by Xpert Carba-R assay were 4 KPC and one each of NDM, VIM, and OXA-48. In all of these cases, positive results were fairly late (threshold cycles [CTs] ranged from 26.2 to 28.2 for KPCs and were >31 for the remaining carbapenemases).
TABLE 5.
Discrepant testing resultsa
| Sample | Reference method |
Target(s) detected by Xpert Carba-R assay | Discrepant analysis |
||
|---|---|---|---|---|---|
| Culture | Sequencing of isolate | DNA extraction and sequencing of MacConkey broth | Outcomeb | ||
| A | No isolate | NAc | IMP-1 | IMP-1 | TP |
| B | Acinetobacter baumannii | No bandsd | KPC | KPC | TP |
| C | Klebsiella pneumoniae | OXA-48 | OXA-48, VIM | OXA-48, VIM | TP |
| D | No isolate | NA | OXA-48 | OXA-48 | TP |
| E | Klebsiella pneumoniae | OXA-48 | OXA-48, VIM | OXA-48, VIM | TP |
| F | No isolate | NA | VIM | VIM | TP |
| G | No isolate | NA | KPC | KPC | TP |
| H | No isolate | NA | OXA-48, VIM | OXA-48, VIM | TP |
| I | No isolate | NA | OXA-48 | OXA-48 | TP |
| J | Enterobacter cloacae | KPC | KPC, NDM | KPC, NDM | TP |
| K | Klebsiella pneumoniae | OXA-48 | OXA-48, VIM | OXA-48, VIM | TP |
| L | No isolate | NA | KPC | No target gene | FP |
| M | No isolate | NA | NDM | No target gene | FP |
| N | No isolate | NA | VIM | No target gene | FP |
| O | No isolate | NA | KPC | No target gene | FP |
| P | No isolate | NA | KPC | No target gene | FP |
| Q | No isolate | NA | KPC | No target gene | FP |
| R | No isolate | NA | OXA-48 | No target gene | FP |
True-positive and false-positive results are shown.
TP, true positive; FP, false positive.
NA, not applicable.
No bands indicate that the sample did not meet PCR product band size inclusion criteria.
DISCUSSION
Detection of fecal carriers of CPO has become a routine clinical practice in many parts of the world and is recommended by public health organizations for the containment of the spread of these isolates (3, 23). Different approaches are used in different countries and institutions, with some culturing rectal swabs on chromogenic media. More recently, molecular approaches have been developed to increase detection sensitivity and decrease reporting time (7, 24). We evaluated, through a prospective multisite study, the Cepheid Xpert Carba-R assay, a PCR-based method in a cartridge format developed for detecting carbapenemase genes in rectal swabs and run on the GeneXpert platform. This assay was previously evaluated, but only three carbapenemase targets (blaKPC, blaNDM, and blaVIM) were included (15). In the current version, two additional targets were incorporated, blaOXA-48 and blaIMP-1, to broaden the scope of the carbapenemases detected. OXA-48 is increasingly recognized in most European countries, predominating in most of the Mediterranean countries, whereas IMP-1 is increasingly detected in Asia and Latin American countries (3, 25).
Overall, 633 specimens, including clinical rectal swabs and contrived samples, were studied, yielding the potential detection of 3,165 carbapenemase targets. Due to the absence or low prevalence of certain carbapenemase genes in organisms found in clinical specimens, such as IMP-1, VIM, or NDM carbapenemases, that reflects the local epidemiology of participating centers, contrived specimens were also included in the evaluation. This approach is normally used in molecular diagnostic platforms for in vitro and clinical evaluations.
For our evaluation, the LODs of different carbapenemase targets were previously calculated (see Materials and Methods) (17). Additionally, analytical reactivity (inclusivity) and potential cross-reactivity (exclusivity) were also calculated, yielding 100% detection of target-containing organisms and no cross-reactions with other resistance genes, respectively (17). Moreover, it is notable that, in our evaluation, we used a highly sensitive method to resolve potential discrepancies between the reference method (culture plus DNA sequencing) and the Cepheid Xpert Carba-R assay. This consisted of DNA extraction and sequencing from the selective enrichment broth (MacConkey with a 10-μg meropenem disk) inoculated with the original specimens. With this approach, overall performance was excellent with sensitivity, specificity, PPV, and NPV all higher than 95%. When performance was evaluated separately for each carbapenemase, the performance was best for IMP-1, and only sensitivities and positive predictive values for VIM and KPC targets were lower than 95%. Unlike other systems for direct detection of carbapenemase genes in rectal swabs, we did not see high false-positive rates for metallo-beta-lactamase targets (26).
Our multisite prospective evaluation was performed for the initial European marketing authorization of the Xpert Carba-R assay and was carried out under the European directive on in vitro diagnostic medical devices (16). A limitation of our evaluation was that the Xpert Carba-R assay included targets for widespread carbapenemases at the time of the evaluation but did not include some of the new emerging OXA-48 variants, such as OXA-181 and OXA-232 carbapenemases. OXA-181 is one of the most prevalent carbapenemases in South Africa and is increasingly recognized in Asia and Europe (27–29), while OXA-232 is also increasingly detected in Asia (30). Recent reports demonstrated that the Xpert Carba-R version evaluated in our study was unable to detect these two variants (31–33). Nevertheless, this was corrected in a novel Xpert Carba-R version recently marketed, which accurately detected these OXA-48 variants (34). The absence of detection of the OXA-181 carbapenemase was also noted with other commercial molecular assays that did not originally include this target. However, as in the Xpert Carba-R assay, subsequent modification of the assay allowed detection of OXA-181 (34).
In our study, with the exception of 6 specimens (Table 2) in which false-negative results were obtained, the Xpert Carba-R assay accurately detected all different targeted carbapenemase genes, including those encoding the KPC, NDM, VIM, IMP-1, and OXA-48 enzymes. The absence of detection in these 6 specimens was not associated with a specific carbapenemase and may be associated with low bacterial load in the rectal swab specimens.
An additional advantage of the Cepheid Xpert Carba-R assay is the ability to detect multiple resistance genes, an outcome not previously reported in other evaluations (31). This occured in 6 cases (Tables 1 and 5). In 4 of them, the reference culture method failed to detect this situation, but the genes were subsequently detected by PCR and DNA sequencing from the MacConkey broth. A potential explanation for these results may be that the organisms recovered by reference culture are present in larger amounts, which would limit the growth of the other organisms present in a lower proportion, or because both carbapenemase-bearing isolates were morphologically identical and one of them was likely missed during the selection of the colonies.
However, a positive result with the Xpert Carba-R assay but negative by culture may happen if the patient had been receiving antibiotics, or the organism may carry a modified sequence of the target gene, which was not expressed or was expressed at low levels.
In conclusion, the Cepheid Xpert Carba-R assay is an accurate technique for the detection of CPO in rectal swab specimens. This assay allows rapid identification of patients colonized with CPO, which can guide infection control programs designed to limit the spread of these organisms in health care settings.
ACKNOWLEDGMENTS
We thank Mona Patel, employee from Cepheid, for technical support during the evaluation.
Funding for this study was provided by Cepheid.
R.C. and P.R.-G. have participated in educational programs supported by Cepheid. The other authors have no conflicts of interest.
REFERENCES
- 1.Glasner C, Albiger B, Buist G, Tambić Andrasević A, Canton R, Carmeli Y, Friedrich AW, Giske CG, Glupczynski Y, Gniadkowski M, Livermore DM, Nordmann P, Poirel L, Rossolini GM, Seifert H, Vatopoulos A, Walsh T, Woodford N, Donker T, Monnet DL, Grundmann H, European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) Working Group. 2013. Carbapenemase-producing Enterobacteriaceae in Europe: a survey among national experts from 39 countries, February 2013. Euro Surveill 18(28):pii=20525 http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20525. [DOI] [PubMed] [Google Scholar]
- 2.World Health Organization. 2014. Antimicrobial resistance: global report on surveillance. World Health Organization, Geneva, Switzerland. [Google Scholar]
- 3.Albiger B, Glasner C, Struelens MJ, Grundmann H, Monnet DL, European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) Working Group. 2015. Carbapenemase-producing Enterobacteriaceae in Europe: assessment by national experts from 38 countries, May 2015. Euro Surveill 20(45):pii=30062 http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=21300. [DOI] [PubMed] [Google Scholar]
- 4.Nordmann P, Poirel L. 2014. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clin Microbiol Infect 20:821–830. doi: 10.1111/1469-0691.12719. [DOI] [PubMed] [Google Scholar]
- 5.Dortet L, Bréchard L, Cuzon G, Poirel L, Nordmann P. 2014. Strategy for rapid detection of carbapenemase-producing Enterobacteriaceae. Antimicrob Agents Chemother 58:2441–2445. doi: 10.1128/AAC.01239-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Hrabák J, Chudáčková E, Papagiannitsis CC. 2014. Detection of carbapenemases in Enterobacteriaceae: a challenge for diagnostic microbiological laboratories. Clin Microbiol Infect 20:839–853. doi: 10.1111/1469-0691.12678. [DOI] [PubMed] [Google Scholar]
- 7.Viau R, Frank KM, Jacobs MR, Wilson B, Kaye K, Donskey CJ, Perez F, Endimiani A, Bonomo RA. 2016. Intestinal carriage of carbapenemase-producing organisms: current status of surveillance methods. Clin Microbiol Rev 29:1–27. doi: 10.1128/CMR.00108-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL. 2012. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev 25:682–707. doi: 10.1128/CMR.05035-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Hornsey M, Phee L, Woodford N, Turton J, Meunier D, Thomas C, Wareham DW. 2013. Evaluation of three selective chromogenic media, CHROMagar ESBL, CHROMagar CTX-M and CHROMagar KPC, for the detection of Klebsiella pneumoniae producing OXA-48 carbapenemase. J Clin Pathol 66:348–350. doi: 10.1136/jclinpath-2012-201234. [DOI] [PubMed] [Google Scholar]
- 10.Nijhuis R, Samuelsen O, Savelkoul P, van Zwet A. 2013. Evaluation of a new real-time PCR assay (Check-Direct CPE) for rapid detection of KPC, OXA-48, VIM, and NDM carbapenemases using spiked rectal swabs. Diagn Microbiol Infect Dis 77:316–320. doi: 10.1016/j.diagmicrobio.2013.09.007. [DOI] [PubMed] [Google Scholar]
- 11.van der Zee A, Roorda L, Bosman G, Fluit AC, Hermans M, Smits PH, van der Zanden AG, Te Witt R, Bruijnesteijn van Coppenraet LE, Cohen Stuart J, Ossewaarde JM. 2014. Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC. BMC Infect Dis 14:27. doi: 10.1186/1471-2334-14-27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.García-Fernández S, Morosini MI, Marco F, Gijón D, Vergara A, Vila J, Ruiz-Garbajosa P, Cantón R. 2015. Evaluation of the eazyplex SuperBug CRE system for rapid detection of carbapenemases and extended-spectrum β-lactamases in clinical Enterobacteriaceae isolates recovered at two Spanish hospitals. J Antimicrob Chemother 70:1047–1050. [DOI] [PubMed] [Google Scholar]
- 13.Cuzon G, Naas T, Bogaerts P, Glupczynski Y, Nordmann P. 2012. Evaluation of a DNA microarray for the rapid detection of extended-spectrum β-lactamases (TEM, SHV and CTX-M), plasmid-mediated cephalosporinases (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and carbapenemases (KPC, OXA-48, VIM, IMP and NDM). J Antimicrob Chemother 67:1865–1869. doi: 10.1093/jac/dks156. [DOI] [PubMed] [Google Scholar]
- 14.Braun SD, Monecke S, Thürmer A, Ruppelt A, Makarewicz O, Pletz M, Reiβig A, Slickers P, Ehricht R. 2014. Rapid identification of carbapenemase genes in Gram-negative bacteria with an oligonucleotide microarray-based assay. PLoS One 9(7):e102232. doi: 10.1371/journal.pone.0102232. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Tenover FC, Canton R, Kop J, Chan R, Ryan J, Weir F, Ruiz-Garbajosa P, LaBombardi V, Persing DH. 2013. Detection of colonization by carbapenemase-producing Gram-negative bacilli in patients by use of the Xpert MDRO assay. J Clin Microbiol 51:3780–3787. doi: 10.1128/JCM.01092-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.European Parliament. 1998. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices. Official J European Communities 331:1–37. [Google Scholar]
- 17.Cepheid. 2014. Xpert Carba-R package insert. Cepheid, Sunnyvale, CA. [Google Scholar]
- 18.Lolans K, Calvert K, Won S, Clark J, Hayden MK. 2010. Direct ertapenem disk screening method for identification of KPC-producing Klebsiella pneumoniae and Escherichia coli in surveillance swab specimens. J Clin Microbiol 48:836–841. doi: 10.1128/JCM.01988-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Clinical and Laboratory Standards Institute. 2014. Performance standards for antimicrobial susceptibility testing: 23rd informational supplement CLSI M100-S24. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 20.Clark RB, Lewinski MA, Loeffelholz MJ, Tibetts RJ. 2009. Cumitech 31A, verification and validation of procedures in the clinical microbiology laboratory. ASM Press, Washington, DC. [Google Scholar]
- 21.Hollander M, Wolfe DA. 1973. Nonparametric statistical methods. John Wiley & Sons, New York, NY. [Google Scholar]
- 22.Fleiss JL, Cohen J, Everitt BS. 1969. Large sample standard errors of kappa and weighted kappa. Psycholog Bull 72:323–327. doi: 10.1037/h0028106. [DOI] [Google Scholar]
- 23.Centers for Disease Control and Prevention (CDC). 2009. Guidance for control of infections with carbapenem-resistant or carbapenemase-producing Enterobacteriaceae in acute care facilities. MMWR Morb Mortal Wkly Rep 58:256–260. [PubMed] [Google Scholar]
- 24.Savard P, Perl TM. 2014. Combating the spread of carbapenemases in Enterobacteriaceae: a battle that infection prevention should not lose. Clin Microbiol Infect 20:854–861. doi: 10.1111/1469-0691.12748. [DOI] [PubMed] [Google Scholar]
- 25.Maya JJ, Ruiz SJ, Blanco VM, Gotuzzo E, Guzman-Blanco M, Labarca J, Salles M, Quinn JP, Villegas MV. 2013. Current status of carbapenemases in Latin America. Expert Rev Anti Infect Ther 11:657–667. doi: 10.1586/14787210.2013.811924. [DOI] [PubMed] [Google Scholar]
- 26.Lau AF, Fahle GA, Kemp MA, Jassem AN, Dekker JP, Frank KM. 2015. Clinical performance of Check-Direct CPE, a multiplex PCR for direct detection of blaKPC, blaNDM and/or blaVIM, and blaOXA-48 from perirectal swabs. J Clin Microbiol 53:3729–3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Balm MN, Ngan G, Jureen R, Lin RT, Teo JW. 2013. OXA-181-producing Klebsiella pneumoniae establishing in Singapore. BMC Infect Dis 13:58. doi: 10.1186/1471-2334-13-58. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Brink AJ, Coetzee J, Corcoran C, Clay CG, Hari-Makkan D, Jacobson RK, Richards GA, Feldman C, Nutt L, van Greune J, Deetlefs JD, Swart K, Devenish L, Poirel L, Nordmann P. 2013. Emergence of OXA-48 and OXA-181 carbapenemases among Enterobacteriaceae in South Africa and evidence of in vivo selection of colistin resistance as a consequence of selective decontamination of the gastrointestinal tract. J Clin Microbiol 51:369–372. doi: 10.1128/JCM.02234-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Kayama S, Koba Y, Shigemoto N, Kuwahara R, Kakuhama T, Kimura K, Hisatsune J, Onodera M, Yokozaki M, Ohge H, Sugai M. 2015. Imipenem-susceptible, meropenem-resistant Klebsiella pneumoniae producing OXA-181 in Japan. Antimicrob Agents Chemother 59:1379–1380. doi: 10.1128/AAC.04330-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Jeong SH, Lee KM, Lee J, Bae IK, Kim JS, Kim HS, Song W. 2015. Clonal and horizontal spread of the blaOXA-232 gene among Enterobacteriaceae in a Korean hospital. Diagn Microbiol Infect Dis 82:70–72. doi: 10.1016/j.diagmicrobio.2015.02.001. [DOI] [PubMed] [Google Scholar]
- 31.Anandan S, Damodaran S, Gopi R, Bakthavatchalam YD, Veeraraghavan B. 2015. Rapid screening for carbapenem resistant organisms: current results and future approaches. J Clin Diagn Res 9:DM01–DM03. doi: 10.7860/JCDR/2015/14246.6530. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Decousser JW, Poirel L, Desroches M, Jayol A, Denamur E, Nordmann P. 2015. Failure to detect carbapenem-resistant Escherichia coli producing OXA-48-like using the Xpert Carba-R assay. Clin Microbiol Infect 21:e9–e10. doi: 10.1016/j.cmi.2014.09.006. [DOI] [PubMed] [Google Scholar]
- 33.Lafeuille E, Laouira S, Sougakoff W, Soulier-Escrihuela O, Leconte J, Garrec H, Tourret J, Jarlier V, Robert J. 2015. Detection of OXA-48-like carbapenemase genes by the Xpert Carba-R test: room for improvement. Int J Antimicrob Agents 45:441–442. doi: 10.1016/j.ijantimicag.2014.12.009. [DOI] [PubMed] [Google Scholar]
- 34.Findlay J, Hopkins KL, Meunier D, 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. [DOI] [PubMed] [Google Scholar]