The immunochromatographic assay NG-Test Carba 5 (NG-Biotech) was evaluated with a collection of 107 carbapenemase-producing nonfermenters (CP-NF) (55 Pseudomonas spp., 51 Acinetobacter spp., and 1 Achromobacter xylosoxidans isolate) and 61 carbapenemase-negative isolates. All KPC, VIM, and NDM carbapenemase producers tested were accurately detected. Of the 16 IMP variants tested, 6 (37.5%) variants were not detected.
KEYWORDS: Acinetobacter, IMP, KPC, NDM, Pseudomonas aeruginosa, VIM, carbapenemases, rapid diagnostic
ABSTRACT
The immunochromatographic assay NG-Test Carba 5 (NG-Biotech) was evaluated with a collection of 107 carbapenemase-producing nonfermenters (CP-NF) (55 Pseudomonas spp., 51 Acinetobacter spp., and 1 Achromobacter xylosoxidans isolate) and 61 carbapenemase-negative isolates. All KPC, VIM, and NDM carbapenemase producers tested were accurately detected. Of the 16 IMP variants tested, 6 (37.5%) variants were not detected. Considering the epidemiology of CP-NFs in France, the NG-Test Carba 5 would detect 89.4% of CP Pseudomonas spp. but only 12.9% of CP Acinetobacter spp.
INTRODUCTION
The production of carbapenemases among Pseudomonas and Acinetobacter species has become a noteworthy mechanism for multidrug resistance. Whereas carbapenem resistance is often associated with nontransferable mechanisms, such as porin (OprD) deficiency in Pseudomonas aeruginosa, most carbapenem-resistant Acinetobacter isolates produce a carbapenemase (1, 2). Carbapenemases belong to three classes (A, B, and D) according to the Ambler classification (3). While class B metallo-β-lactamases of the VIM type and IMP type are predominant in P. aeruginosa isolates, carbapenem-hydrolyzing class D β-lactamases (CHDL) of the OXA-23-, OXA-24/40, OXA-58, and OXA-143 groups are still the most prevalent carbapenem resistance determinants in Acinetobacter baumannii, but NDM producers are increasingly being reported worldwide (2, 4–6). In contrast, KPC ambler class A enzymes and OXA-48-like β-lactamases remain relatively uncommon in nonfermentative Gram-negative species (7–9). In this context, the rapid detection of carbapenemase-producing bacteria has become crucial to prevent their dissemination.
Recently, the NG-Test Carba 5 immunochromatographic assay (ICA) (NG Biotech, Guipry, France) was developed to detect the five most widespread carbapenemase families in carbapenemase-producing Enterobacterales (CPEs) (i.e., KPC-, NDM-, VIM-, IMP-, and OXA-48-like enzymes). It was demonstrated to accurately identify the claimed enzymes (10). Compared with other ICAs developed to detect CPEs (RESIT-4 O.K.V.N.; Coris BioConcept, Belgium), the NG-Test Carba 5 targets in addition the very heterogeneous family of IMP-type enzymes, which are more prevalent in nonfermenters than in Enterobacterales (11). Here, we have evaluated the performance of the NG-Test Carba 5 for the detection of carbapenemase-producing Pseudomonas spp. and Acinetobacter spp. encountered in France.
A collection of 168 nonfermenters with PCR-characterized β-lactamase content was used to evaluate the ICA NG-Test Carba 5. Those strains were identified to the species level using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MALDI Biotyper CA system; Bruker Daltonics, Billerica, MA, USA) and had previously been characterized at the molecular level for their carbapenemase content (12, 13). This collection included 105 Pseudomonas spp. (P. aeruginosa, n = 96; P. putida, n = 4; P. stutzeri, n = 2; P. fluorescens, n = 1; P. alcaligenes n = 1; P. otitidis n = 1), 62 Acinetobacter spp. (A. baumannii, n = 56; A. pittii, n = 3; A. junii, n = 1; Acinetobacter genomosp. 16, n = 1; A. nosocomialis, n = 1), and 1 Achromobacter xylosoxidans strain. Among these 168 isolates, 107 were carbapenemase producers (55 Pseudomonas spp., 51 Acinetobacter spp., and 1 A. xylosoxidans strain) and 61 were not carbapenemase producers (50 Pseudomonas spp. and 11 Acinetobacter spp.). The produced carbapenemases included 3 KPC enzymes and 71 metallo-β-lactamases targeted by the NG-Test Carba 5 (22 VIM, 24 IMP, and 25 NDM enzymes). Thirty-three isolates produced a carbapenemase not targeted by the NG-Test Carba 5 (4 of the GES type, 5 OXA-23 type, 7 OXA-24/40 type, 7 OXA-58 type, 2 GIM-1, 2 AIM-1, 1 SPM-1, 1 DIM-1, 1 PAM-1, 1 POM-1, 1 OXA-198, and 1 SIM-1). According to the EUCAST guidelines (14), the majority of the carbapenemase-producing strains were not susceptible to imipenem (101/107, 94.4%) or to meropenem (102/107, 95.3%) (see Table S1 in the supplemental material).
The NG-Test Carba 5 identified all KPC (n = 3) and VIM (n = 22) producers, with no false-positive results. Among the NDM-positive isolates, 92% (n = 23/25) were correctly identified. The two isolates that yielded negative results after 15 min of incubation (manufacturer’s recommendations) became slightly positive after 30 min. The test allowed the detection of 62.5% of the IMP producers (n = 15/24). As has already been reported, the detection of IMP-type carbapenemases remains challenging considering the high sequence diversity within the IMP family (15). Here, 10 out of the 16 different IMP variants were correctly detected. The false-negative results correspond to the IMP-13 clade (IMP-13 and IMP-37), IMP-15, the IMP-18 clade (IMP-18 and IMP-71), and IMP-63 (Table 1, Fig. S1). All 61 non-carbapenemase-producing isolates and all the 33 isolates producing a carbapenemase different from those targeted by the NG-Test Carba 5 gave negative test results, demonstrating no cross-reactivity between OXA CHDLs encountered in Acinetobacter (OXA-23, -24/-40, and -58) and OXA-48-like enzymes identified in Enterobacterales.
TABLE 1.
Results of the NG-Test Carba 5 on the collection of Pseudomonas spp. and Acinetobacter spp.a
| Category | Species | Carbapenemase | No. of isolates | NG-Test Carba 5 result for strains with: |
% sensitivity (95% CI) | % specificity (95% CI) | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| NDM | IMP | VIM | OXA | KPC | ||||||
| Carbapenemase producers | ||||||||||
| KPC type | P. aeruginosa | KPC-2 | 3 | N | N | N | N | P | 100 (31.0–100) | 100 (97.2–100) |
| GES type | P. aeruginosa | GES-2 | 1 | N | N | N | N | N | ||
| P. aeruginosa | GES-5 | 1 | N | N | N | N | N | |||
| P. aeruginosa | GES-6 | 1 | N | N | N | N | N | |||
| A. baumannii | GES-14 | 1 | N | N | N | N | N | |||
| VIM type | A. xylosoxidans | VIM-1 | 1 | N | N | P | N | N | 100 (81.5–100) | 100 (96.8–100) |
| P. aeruginosa | VIM-1 | 1 | N | N | P | N | N | |||
| P. fluorescens | VIM-2 | 1 | N | N | P | N | N | |||
| P. stutzeri | VIM-2 | 1 | N | N | P | N | N | |||
| P. putida | VIM-2 | 1 | N | N | P | N | N | |||
| P. aeruginosa | VIM-2 | 9 | N | N | P | N | N | |||
| P. aeruginosa | VIM-4 | 1 | N | N | P | N | N | |||
| Acinetobacter genomosp. 16 | VIM-4 | 1 | N | N | P | N | N | |||
| A. pittii | VIM-4 | 1 | N | N | P | N | N | |||
| P. aeruginosa | VIM-6 | 1 | N | N | P | N | N | |||
| P. aeruginosa | VIM-11 | 1 | N | N | P | N | N | |||
| P. aeruginosa | VIM-28 | 1 | N | N | P | N | N | |||
| P. aeruginosa | VIM-30 | 1 | N | N | P | N | N | |||
| P. putida | VIM-60 | 1 | N | N | P | N | N | |||
| IMP type | P. stutzeri | IMP-1 | 1 | N | P | N | N | N | 62.5 (40.8–80.4) | 100 (96.8–100) |
| P. putida | IMP-1 | 1 | N | P | N | N | N | |||
| P. aeruginosa | IMP-1 | 1 | N | P | N | N | N | |||
| A. baumannii | IMP-1 | 1 | N | P | N | N | N | |||
| A. pittii | IMP-1 | 1 | N | P | N | N | N | |||
| P. aeruginosa | IMP-2 | 1 | N | P | N | N | N | |||
| A. baumannii | IMP-4 | 1 | N | P | N | N | N | |||
| P. aeruginosa | IMP-5 | 1 | N | P | N | N | N | |||
| A. pittii | IMP-5 | 1 | N | P | N | N | N | |||
| P. aeruginosa | IMP-10 | 1 | N | P | N | N | N | |||
| P. aeruginosa | IMP-13 | 3 | N | N | N | N | N | |||
| P. aeruginosa | IMP-15 | 1 | N | N | N | N | N | |||
| P. aeruginosa | IMP-18 | 1 | N | N | N | N | N | |||
| P. aeruginosa | IMP-19 | 1 | N | P | N | N | N | |||
| P. aeruginosa | IMP-26 | 1 | N | P | N | N | N | |||
| P. aeruginosa | IMP-29 | 1 | N | P | N | N | N | |||
| P. aeruginosa | IMP-39 | 1 | N | P | N | N | N | |||
| P. putida | IMP-63 | 1 | N | N | N | N | N | |||
| P. aeruginosa | IMP-63 | 1 | N | N | N | N | N | |||
| P. aeruginosa | IMP-71 | 1 | N | N | N | N | N | |||
| P. aeruginosa | IMP-79 | 1 | N | P | N | N | N | |||
| NDM type | P. aeruginosa | NDM-1 | 2 | P | N | N | N | N | At 15 min, 92.0 (72.4–98.6); at 30 min, 100 (83.4–100) | At 15 min, 100 (96.8–100); at 30 min, 100 (96.8–100) |
| A. baumannii | NDM-1 | 13 | P | N | N | N | N | |||
| A. baumannii | NDM-1 | 2 | Nb | N | N | N | N | |||
| A. baumannii | NDM-2 | 1 | P | N | N | N | N | |||
| A. baumannii | NDM-9 | 1 | P | N | N | N | N | |||
| OXA type | P. aeruginosa | OXA-198 | 1 | N | N | N | N | N | ||
| A. baumannii | OXA-23 | 5 | N | N | N | N | N | |||
| A. baumannii | OXA-25 | 1 | N | N | N | N | N | |||
| A. baumannii | OXA-26 | 1 | N | N | N | N | N | |||
| A. baumannii | OXA-72 | 5 | N | N | N | N | N | |||
| A. baumannii | OXA-58 | 4 | N | N | N | N | N | |||
| A. baumannii | OXA-92 | 1 | N | N | N | N | N | |||
| A. baumannii | OXA-97 | 1 | N | N | N | N | N | |||
| A. nosocomialis | OXA-420 | 1 | N | N | N | N | N | |||
| Other-carbapenemaseproducers | P. aeruginosa | GIM-1 | 2 | N | N | N | N | N | ||
| P. aeruginosa | AIM-1 | 2 | N | N | N | N | N | |||
| P. aeruginosa | SPM-1 | 1 | N | N | N | N | N | |||
| P. aeruginosa | DIM-1 | 1 | N | N | N | N | N | |||
| A. baumannii | SIM-1 | 1 | N | N | N | N | N | |||
| P. otitidis | POM-1 | 1 | N | N | N | N | N | |||
| P. alcaligenes | PAM-1 | 1 | N | N | N | N | N | |||
| Multiple-carbapenemase producers | A. baumannii | OXA-23 + NDM-1 | 6 | P | N | N | N | N | ||
| A. junii | IMP-37 + OXA-58 | 1 | N | N | N | N | N | |||
| Non-carbapenemase producers | P. aeruginosa | 50 | N | N | N | N | N | |||
| A. baumannii | 11 | N | N | N | N | N | ||||
P, positive; N, negative; 95% CI, 95% confidence interval, calculated using online VassarStats software (http://vassarstats.net/).
The NDM band was visible at 30 min but not at 15 min.
In countries where the epidemiology of carbapenemase-positive P. aeruginosa strains is largely dominated by VIM producers and where the incidence of IMP producers is quite low (8% in 2017 in France [P. Plésiat, unpublished results]), it seems reasonable to recommend the use of the NG-Test Carba 5. Considering the French epidemiology of CP P. aeruginosa strains in 2017, the NG-Test Carba 5 might have correctly detected 89.4% of them. However, phenotypic methods (e.g., carbapenem hydrolysis tests, such as the RAPIDEC Carba NP) are still currently required for strains highly suspected of producing a metallo-β-lactamase (carbapenem resistance and high-level resistance to ceftolozane-tazobactam) that produce a negative result by the current version of the NG-Test Carba 5. Of note, the future version of the NG-Test Carba 5 that will be commercialized in 2019 will include all these undetected IMP variants (the IMP-13 clade [IMP-13 and IMP-37], IMP-14, IMP-15, the IMP-18 clade [IMP-18 and IMP-71], and IMP-63 [L. Dortet, unpublished results]), which might be helpful for the accurate detection of carbapenemase producers in countries where IMP producers are more prevalent (e.g., Japan, South Korea, and Taiwan).
In summary, the NG-Test Carba 5 is a useful tool for the accurate identification of CP Pseudomonas spp. Since the most prevalent carbapenemases identified in Acinetobacter spp. are not targeted by the assay, implementation of this test to identify CP Acinetobacter spp. is not recommended or should at least be combined with the OXA-23 K-Set ICA from Coris BioConcept (16).
Supplementary Material
ACKNOWLEDGMENTS
This work was partially supported by the French Ministry of Health via the Santé Publique France Agency and by the University Paris-Sud, France, and Assistance Publique des Hôpitaux de Paris, Paris, France.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00968-19.
REFERENCES
- 1.Simner PJ, Johnson JK, Brasso WB, Anderson K, Lonsway DR, Pierce VM, Bobenchik AM, Lockett ZC, Charnot-Katsikas A, Westblade LF, Yoo BB, Jenkins SG, Limbago BM, Das S, Roe-Carpenter DE. 2018. Multicenter evaluation of the modified carbapenem inactivation method and the Carba NP for detection of carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter baumannii. J Clin Microbiol 56:e01369-17. doi: 10.1128/JCM.01369-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Potron A, Poirel L, 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. [DOI] [PubMed] [Google Scholar]
- 3.Bush K. 2013. Carbapenemases: partners in crime. J Glob Antimicrob Resist 1:7–16. doi: 10.1016/j.jgar.2013.01.005. [DOI] [PubMed] [Google Scholar]
- 4.Oliver A, Mulet X, López-Causapé C, Juan C. 2015. The increasing threat of Pseudomonas aeruginosa high-risk clones. Drug Resist Updat 21-22:41–59. doi: 10.1016/j.drup.2015.08.002. [DOI] [PubMed] [Google Scholar]
- 5.Evans BA, Amyes SG. 2014. OXA beta-lactamases. Clin Microbiol Rev 27:241–263. doi: 10.1128/CMR.00117-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Khan AU, Maryam L, Zarrilli R. 2017. Structure, genetics and worldwide spread of New Delhi metallo-beta-lactamase (NDM): a threat to public health. BMC Microbiol 17:101. doi: 10.1186/s12866-017-1012-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Martinez T, Martinez I, Vazquez GJ, Aquino EE, Robledo IE. 2016. Genetic environment of the KPC gene in Acinetobacter baumannii ST2 clone from Puerto Rico and genomic insights into its drug resistance. J Med Microbiol 65:784–792. doi: 10.1099/jmm.0.000289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Hagemann JB, Pfennigwerth N, Gatermann SG, von Baum H, Essig A. 2018. KPC-2 carbapenemase-producing Pseudomonas aeruginosa reaching Germany. J Antimicrob Chemother 73:1812–1814. doi: 10.1093/jac/dky105. [DOI] [PubMed] [Google Scholar]
- 9.Borah VV, Saikia KK, Hazarika NK. 2016. First report on the detection of OXA-48 beta-lactamase gene in Escherichia coli and Pseudomonas aeruginosa co-infection isolated from a patient in a tertiary care hospital in Assam. Indian J Med Microbiol 34:252–253. doi: 10.4103/0255-0857.176842. [DOI] [PubMed] [Google Scholar]
- 10.Boutal H, Vogel A, Bernabeu S, Devilliers K, Creton E, Cotellon G, Plaisance M, Oueslati S, Dortet L, Jousset A, Simon S, Naas T, Volland H. 2018. A multiplex lateral flow immunoassay for the rapid identification of NDM-, KPC-, IMP- and VIM-type and OXA-48-like carbapenemase-producing Enterobacteriaceae. J Antimicrob Chemother 73:909–915. doi: 10.1093/jac/dkx521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Greissl C, Saleh A, Hamprecht A. 2019. Rapid detection of OXA-48-like, KPC, NDM, and VIM carbapenemases in Enterobacterales by a new multiplex immunochromatographic test. Eur J Clin Microbiol Infect Dis 38:331–335. doi: 10.1007/s10096-018-3432-2. [DOI] [PubMed] [Google Scholar]
- 12.Dortet L, Boulanger A, Poirel L, Nordmann P. 2014. Bloodstream infections caused by Pseudomonas spp.: how to detect carbapenemase producers directly from blood cultures. J Clin Microbiol 52:1269–1273. doi: 10.1128/JCM.03346-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Dortet L, Poirel L, Errera C, Nordmann P. 2014. CarbAcineto NP test for rapid detection of carbapenemase-producing Acinetobacter spp. J Clin Microbiol 52:2359–2364. doi: 10.1128/JCM.00594-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.EUCAST. 2019. Clinical breakpoints for bacteria. EUCAST; http://www.eucast.org/clinical_breakpoints/. [Google Scholar]
- 15.Hopkins KL, Meunier D, Naas T, Volland H, Woodford N. 2018. Evaluation of the NG-Test CARBA 5 multiplex immunochromatographic assay for the detection of KPC, OXA-48-like, NDM, VIM and IMP carbapenemases. J Antimicrob Chemother 73:3523–3526. doi: 10.1093/jac/dky342. [DOI] [PubMed] [Google Scholar]
- 16.Riccobono E, Bogaerts P, Antonelli A, Evrard S, Giani T, Rossolini GM, Glupczynski Y. 2019. Evaluation of the OXA-23 K-SeT immunochromatographic assay for the rapid detection of OXA-23-like carbapenemase-producing Acinetobacter spp. J Antimicrob Chemother 74:1455–1457. doi: 10.1093/jac/dkz001. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.