Related articles in EMJ
Performance of Xpert Carba-R Assay for Identification of Carbapenemase Gene in the Clinical Microbiology Laboratory
Department of Laboratory Medicine, Ewha Womans University College of Medicine, Seoul, 1Sejong Hospital, Bucheon, 2Hankook General Hospital, Seoul, KoreaCorrespondence to:
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ewha Med J 2020; 43(3): 39-42
Published July 31, 2020
Copyright © Ewha Womans University School of Medicine.
Methods: The analytical limit of detection was determined with two suspensions of carbapenemase-producing Enterobacteriaceae (CPE) isolates (KPC and NDM). A total of 52 specimens were evaluated: 21 bacterial isolates from clinical specimens, 21 rectal swabs, and 10 contrived stool specimens.
Results: In bacterial isolates, concordant results between the Xpert Carba-R Assay and PCR were found in 20 of 21; 8 KPC, 8 NDM, 1 IMP, and 2 multiple carbapenamase genes (KPC/NDM, NDM/OXA) were detected both by Xpert Carba-R Assay and PCR. In one GES-positive isolate, Xpert Carba-R Assay showed a negative result as expected. One VIM-positive isolate tested negative by Xpert Carba-R Assay. Complete concordance was seen in rectal swab specimens: 4 specimens with KPC and 17 specimens with negative results both by Xpert Carba-R Assay and surveillance culture. Among the 10 contrived stool specimens, Xpert Carba-R Assay detected carbapenemase genes in 9 specimens as expected according to the CPE strains spiked into the contrived stool; 2 KPC, 4 NDM, 1 IMP, and 2 multiple carabapenamase genes (NDM/KPC, NDM/OXA). One VIM-positive specimen tested negative by Xpert Carba-R Assay.
Conclusion: In conclusion, the Xpert Carba-R Assay can be used to identify carbapenemase gene in bacterial isolates cultured from clinical specimens and detect CPE carrier using rectal swab in clinical laboratories.
Rapid detection of carbapenemase-producing
To determine the analytical limit of detection of the assay, 2 suspensions of
A total of 52 specimens were evaluated to assess the reproducibility of the assay: 21 bacterial isolates from clinical specimens, 21 rectal swabs, and 10 contrived stool specimens (Table 1). Bacterial isolates from clinical specimens were retrieved from CPE previously proven by culture, PCR and sequencing. Carbapenemase production was confirmed by PCR and sequencing of carbapenemase genes including
The Xpert Carba-R Assay was run on the GeneXpert platform as per the manufacturer’s instructions, and the results were compared to previously performed culture and/or sequencing results.
This study was considered exempt from institutional review board oversight (EUMC 2018-06-029).
The Xpert Carba-R Assay detected KPC and NDM to a final concentration of 1.5×104 CFU/mL, with threshold cycle 33 for KPC, and 32 for NDM. Gene amplification of KPC and NDM were occurred at the detection limit concentration of 1.5×103 CFU/mL, but were reported negative because the threshold cycle value were outside the effective range.
Overall, concordant results were shown in 50 out of 52 samples (96.2%; Kappa value, 0.92; 95% confidence interval [CI], 0.81 to 1.00). In bacterial isolates, concordant results between the Xpert Carba-R Assay and PCR were found in 20 of 21. Nineteen CPE isolates were detected both by the Xpert Carba-R Assay and PCR: 8 KPC-2, 7 NDM-1, 1 NDM-3, 1 IMP-1, 2 CPE with multiple carbapenemase genes (KPC-2/NDM-1, NDM-5/OXA-181). In one GES-producing isolate, the Xpert Carba-R Assay showed a negative result as expected. One VIM-2-producing isolate tested negative by the Xpert Carba-R Assay. Complete concordance was seen in rectal swab specimens (21 out of 21): 4 specimens with KPC-1, and 17 specimens with negative results both by the Xpert Carba-R Assay and surveillance culture. Among the 10 contrived stool specimens, the Xpert Carba-R Assay detected carbapenemase genes in 9 specimens as expected according to the CPE strains spiked into the contrived stool: 2 KPC-2, 3 NDM-1, 1 NDM-3, 1 IMP-1, and 2 multiple carbapenamases specimens (KPC-2/NDM-1, NDM-5/OXA-181). One VIM-2-positive contrived stool specimen tested negative by the Xpert Carba-R Assay. In conclusion, there were two discrepant results due to the one VIM-2-producing isolate, one from the bacterial isolate test and the other from the contrived stool specimen test (Table 1).
In this study, we verified the performance of the Xpert Carba-R Assay to introduce into routine use in the clinical microbiology laboratory and it showed complete concordance with comparable methods for major carbapenemases except VIM. The assay showed good performance for all bacterial isolates, rectal swab and contrived stool specimen, and we decided to use the assay for carbapenemase identification in the bacterial isolates cultured from clinical specimens and CPE carrier detection using rectal swab.
The limit of detection of this study was higher than that for the KPC/NDM-producing standard strain provided by the manufacturer  and also higher compared with data published in other studies . The limit of detection may be increased according to the concentration of the resistant gene in the isolate. Clinical isolates were used in this study and the burden of gene in bacteria is difficult to measure.
Several studies have evaluated the performance of the assay for the bacterial isolates and/or rectal swabs [3,6-9]. Despite the limitations and variations of these studies, the overall Xpert Carba-R Assay showed excellent performance; 96.6% to 100% of sensitivity and 95.3% to 99.13% of specificity.
OXA-181, which is one of the OXA-48 variants, was detected as OXA-48 positive. Previously, the Xpert Carba-R Assay failed to detect the new emerging OXA-48 variants, such as OXA-181 and OXA-232 carbapenemases [10-13]. However, this poor performance was improved in a novel version of the assay, which accurately detected the OXA-48 variants [3-6]. OXA-181 is one of the most prevalent carbapenemases in South Africa and is increasingly recognized in Asia and Europe [14-16].
Our study included one VIM-2-producing
The Xpert Carba-R Assay has several limitations. Most of all, it is not for bacterial identification. Detection of these gene sequences does not indicate the presence of viable organisms. Therefore, when the assay is performed on rectal swab specimens from patients, concomitant cultures are necessary to recover organisms for epidemiological typing, antimicrobial susceptibility testing, and for further confirmatory bacterial identification.
Due to the absence or low prevalence of IMP-1, VIM, and OXA carbapenemases in isolates found in clinical specimens in our laboratory, only one isolate of each carbapenemase was included in this study. And we intentionally included more positive rectal swab specimens than negative samples, consequentially the prevalence of carbapenemases (4/21, 19.0%) was higher than that of the routine surveillance culture in our hospital. The performance of assays depends largely on the local prevalence; in low prevalence setting, false-positive results could be higher.
In conclusion, the Xpert Carba-R Assay can be used to identify carbapenemase gene in bacterial isolates cultured from clinical specimens and detect CPE carrier using rectal swab in clinical laboratories. It can allow the earlier implementation of appropriate treatment and infection control as it is less labor-intensive and yields reliable results quickly.
This work was supported by Ewha Womans University Research Grant of 2016 and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)(No. 2017R1C1B5017422). GeneX provided the reagents for this study but did not involve in either data collection or preparation of manuscript.
- Centers for Disease Control and Prevention. Facility guidance for control of carbapenem-resistant Enterobacteriaceae (CRE) [Internet]. Atlanta: Centers for Disease Control and Prevention; 2015 [cited 2020 Jul 10]. Available from: https://www.cdc.gov/hai/pdfs/cre/cre-guidance-508.pdf.
- Cepheid. Xpert Carba-R package insert. Sunnyvale: Cepheid; 2016.
- Traczewski MM, Carretto E, Canton R, Moore NM; Carba-R Study Team. Multicenter evaluation of the Xpert Carba-R assay for detection of carbapenemase genes in gram-negative isolates.
J Clin Microbiol2018;56:e00272-18.
- Centers for Disease Control and Prevention. Laboratory protocol for detection of carbapenem-resistant or carbapenemase-producing,
Klebsiellaspp. and E. colifrom rectal swabs. Atlanta: Centers for Disease Control and Prevention; 2009.
- Viau R, Frank KM, Jacobs MR, Wilson B, Kaye K, Donskey CJ, et al. Intestinal carriage of carbapenemase-producing organisms: current status of surveillance methods.
Clin Microbiol Rev2016;29:1-27.
- Dortet L, Fusaro M, Naas T. Improvement of the Xpert Carba-R Kit for the detection of carbapenemase-producing enterobacteriaceae.
Antimicrob Agents Chemother2016;60:3832-3837.
- Tato M, Ruiz-Garbajosa P, Traczewski M, Dodgson A, McEwan A, Humphries R, et al. Multisite evaluation of cepheid Xpert Carba-R assay for detection of carbapenemase-producing organisms in rectal swabs.
J Clin Microbiol2016;54:1814-1819.
- Hoyos-Mallecot Y, Ouzani S, Dortet L, Fortineau N, Naas T. 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 Agents2017;49:774-777.
- Moore NM, Canton R, Carretto E, Peterson LR, Sautter RL, Traczewski MM, et al. Rapid identification of five classes of carbapenem resistance genes directly from rectal swabs by use of the Xpert Carba-R assay.
J Clin Microbiol2017;55:2268-2275.
- Lafeuille E, Laouira S, Sougakoff W, Soulier-Escrihuela O, Leconte J, Garrec H, et al. Detection of OXA-48-like carbapenemase genes by the Xpert Carba-R test: room for improvement.
Int J Antimicrob Agents2015;45:441-442.
- Findlay J, Hopkins KL, Meunier D, Woodford N. Evaluation of three commercial assays for rapid detection of genes encoding clinically relevant carbapenemases in cultured bacteria.
J Antimicrob Chemother2015;70:1338-1342.
- Decousser JW, Poirel L, Desroches M, Jayol A, Denamur E, Nordmann P. Failure to detect carbapenem-resistant
Escherichia coliproducing OXA-48-like using the Xpert Carba-R assay. Clin Microbiol Infect2015;21:e9-e10.
- Anandan S, Damodaran S, Gopi R, Bakthavatchalam YD, Veeraraghavan B. Rapid screening for carbapenem resistant organisms: current results and future approaches.
J Clin Diagn Res2015;9:DM01-DM03.
- Kayama S, Koba Y, Shigemoto N, Kuwahara R, Kakuhama T, Kimura K, et al. Imipenem-susceptible, meropenem-resistant
Klebsiella pneumoniaeproducing OXA-181 in Japan. Antimicrob Agents Chemother2015;59:1379-1380.
- Brink AJ, Coetzee J, Corcoran C, Clay CG, Hari-Makkan D, Jacobson RK, et al. Emergence of OXA-48 and OXA-181 carbapenemases among
Enterobacteriaceaein South Africa and evidence of in vivo selection of colistin resistance as a consequence of selective decontamination of the gastrointestinal tract. J Clin Microbiol2013;51:369-372.
- Balm MN, Ngan G, Jureen R, Lin RT, Teo JW. OXA-181-producing
Klebsiella pneumoniaeestablishing in Singapore. BMC Infect Dis2013;13:58.