27 February 2019

Evaluation of the Amplidiag CarbaR+MCR Kit for Accurate Detection of Carbapenemase-Producing and Colistin-Resistant Bacteria

LETTER

As carbapenemase-producing Gram-negative bacilli (CP-GNB) coproducing MCR-1 are now emerging and, thus, compromising the use of last-resort antibiotics (carbapenems and colistin), there is an urgent need for accurate and fast diagnostic tests (14). We have previously shown that the Amplidiag CarbaR+VRE assay displayed a high sensitivity and specificity on colonies of CP-GNB but failed to detect GES carbapenemase-producing organisms (GES-CPOs), which represent 11% of the carbapenemase-producing Pseudomonas aeruginosa (CP-Pa) in France (5). Moreover, this assay was not validated on clinical samples, such as rectal swabs.
Here, we have evaluated an updated assay, the Amplidiag Carba-R+MCR assay, which is a multiplex nucleic acid-based in vitro diagnostic test intended for the detection of CP-GNB, including GES carbapenemase-producing organisms (GES-CPOs) and colistin resistance gene mcr-1/2 from rectal swabs and from cultured colonies. The assay was evaluated during a retrospective evaluation on colonies of 215 GNB with well-characterized resistance mechanisms, during a prospective study on 51 consecutive enterobacterial isolates with reduced susceptibility to carbapenems referred to the French National Reference Center, and on extracted DNA from 100 rectal swabs (including 40 positives) sent to the hygiene unit for CP-GNB screening. This assay was used as recommended by the manufacturer (Mobidiag, Paris, France).
The Amplidiag CarbaR+MCR efficiently detected the big 5 carbapenemase families (KPC, including novel variants tested, such as KPC-5, KPC-6, KPC-12, KPC-14, and KPC-28; NDM, including NDM-4, NDM-5, NDM-6, NDM-7, and NDM-9; VIM, including VIM-4, VIM-9, and VIM-19; IMP, including IMP-8, IMP-11, and IMP-13; OXA-48-like, including OXA-162, OXA-181, OXA-232, and OXA-244) and the main acquired carbapenem-hydrolyzing oxacillinases from Acinetobacter baumannii (OXA-23, OXA-24/OXA-40, OXA-58, and the overexpressed OXA-51-like β-lactamase) in a similar manner to the Amplidiag CarbaR+VRE assay (Table 1). The sensitivity for all carbapenemases in cultured colonies was 99.2%; only one GES-CPO (GES-2-producing P. aeruginosa) was not detected. Overall, the specificity of detection of GES carbapenemases was not optimal since GES noncarbapenemase variants were also detected but with lower threshold cycle (CT) values. The three GES-7 producers have been falsely detected as carbapenemase producers. Indeed, the β-lactamase GES-7 does not host the mutation (Gly170Ser) conferring carbapenemase activity; its spectrum of hydrolysis is restricted to expended-spectrum cephalosporins (Table 1). However, CT values at 23 to 27 were obtained for the extended-spectrum β-lactamase (ESBL) GES-7, unlike GES-CPOs (GES-5 and GES-6) that displayed lower CT values of 12 to 17 (Table 1). Among the GES non-CPOs, the specificity was 38.5%, lowering the specificity of carbapenemase detection to 86.2% (Table 1). Plasmid-encoded colistin resistance genes mcr-1 and mcr-2 were perfectly detected (100% sensitivity; Table 1). As claimed by the manufacturer, MCR variants other than MCR-1 and -2 (here, MCR-3, -3.2, -4, and -5 [6] were not detected; 100% specificity; Table 1).
TABLE 1
TABLE 1 Global performances of the Amplidiag Carba+MCR kit on Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp. colonies grown on MH agar isolates with decreased susceptibility to carbapenems or to colistina
Organism(s) (no. of isolates)β-lactam- or colistin-resistance mechanismAmplidiag Carba+MCR CT valuesb
KPC, NDM, VIM, IMP, OXA-48-like, AcOXAGESMCR
Decreased susceptibility to carbapenems (n = 187)    
    Non-GES/Non-carbapenemase/Nontargeted carbapenemase producers (45)    
        Escherichia coli (2), E. cloacae (4), Enterobacter asburiae (1), Citrobacter freundii (1), Morganella morganii (1), Hafnia alvei (2), Serratia marcescens (2), Klebsiella oxytoca (1), Klebsiella pneumoniae (3), P. aeruginosa (17), A. baumannii (11)↗↗↗ Casec, ↗↗↗ Pase, CTX-M-15, TEM, SHV, OXA-163, OXA-405, PER-1, VEB-1, OXA-32, ↗↗↗ Case, OprD deficiency, Efflux, IMI, NmcA, GIM, AIM, SPM, DIM, OXA-198, None, PER-1, VEB-1, SCO-1, RTG-4, OXA-10, OXA-21, OXA-69, SIM, OXA-143, OXA-253-d--
    Non-GES other carbapenemase producers (113)    
        Enterobacteriaceae (53), P. aeruginosa (30), A. baumannii (30)KPC, NDM, VIM, IMP, OXA-48-like, ISAba1-OXA-51, OXA23, OXA-40, OXA-5810–20--
    GES non-CPO (13)    
        K. pneumoniae (1)GES-1---
        A. baumannii (2)GES-11, GES-12 + OXA-51+ISAba114--
        P. aeruginosa (2)GES-1, GES-9---
        Citrobacter amalonaticus (1), E. cloacae (1), K. oxytoca (1)GES-7-23–27-
        A. baumannii (5)GES-11/GES-12, + OXA-23, + ISAba1-OXA-5111–1626–32-
    GES CPO (16)    
        P. aeruginosa (1)GES-2---
        E. cloacae (5), K. pneumoniae (2), Citrobacter braakii (1), Citrobacter youngae (1)GES-5, GES-6-12–17-
        P. aeruginosa (2)GES-5-14-
        A. baumannii (4)GES-14-14–17-
Decreased susceptibility to colistin (n = 28)    
    Non-/Non-targeted-MCR producers (15)   
        E. coli (4), K. pneumoniae (1), Salmonella spp. (4)ΔpmrBe, ΔmgrB, ND---
        E. coli (4), Salmonella spp. (2)MCR-3, MCR-3.2, MCR-4, MCR-5---
    Targeted MCR producers (13)    
        E. coli (4), Salmonella spp. (3), K. pneumoniae (3)MCR-1, MCR-2--12–19
        E. coli (3)MCR-1 + OXA-48, + NDM-114–16-12–14
a
Sensitivity for carbapenemases, 99.2 % (95% confidence interval [CI], 95.1%–100%); sensitivity for GES-CPO (15/16), 93.7%; for mcr-1/-2, 100% (95% CI, 71.6%–100%); specificity for carbapenemases, 86.2% (95% CI, 74.1%–93.4%); specificity among GES non-CPO (5/13), 38.5 %; for mcr-1/-2, 100% (95% CI, 74.6%–100%).
b
Number of isolates tested. Dark gray boxes represent discrepant results. CT values were rounded up.
c
↗↗↗ Case, abbreviation for overexpressed cephalosporinase.
d
No amplification.
e
Δ, mutation or deletion; ND, not determined.
DNA was extracted from rectal swabs, and the sensitivity was 92.5%; two NDM producers and one OXA-48 producer were not detected by the Amplidiag CarbaR+MCR. These 3 samples contained low concentrations of bacteria, requiring an overnight enrichment step in brain heart infusion medium supplemented with 0.5 µg/ml ertapenem for detection of two of them (Table 2). Conversely, one positive result was obtained with an AcOXA gene in a sample containing a cultivable OXA-48 producing Enterobacter cloacae isolate. In-house PCR confirmed the presence of the blaOXA-23 gene in the DNA extract, despite the absence of Acinetobacter spp. in the culture (7, 8). Accordingly, the global performances of the Amplidiag Carba-R+MCR assay were high (92 to 100% sensitivity and 86 to 100% specificity). Moreover, it can provide a result not only from colonies growing on Mueller-Hinton agar (MHA) or on selective screening medium but also from DNA extracted from clinical rectal swabs in less than three hours (1 h of DNA extraction and less than 2 h for PCR). The Amplidiag Carba-R+MCR assay is well adapted to the French epidemiology of CP-GNB, as it is able to detect 99.57% of CPEs (missing 13 IMI producers and one FRI-1 producer), 100% of CP A. baumannii (CP-Abs), and now 100% of CP-Pas (including the 11% of GES variants that were missed by the previous Carba-R+VRE assay) isolated over the period from 2012 to 2016 in France (9).
TABLE 2
TABLE 2 Global performances of the Amplidiag Carba+MCR kit on DNA extracted from clinical rectal swabsa
Culture on ChromID Carba SMART medium (no. of isolates tested)CarbapenemaseaAmplidiag Carba+MCR CT valuesb
Direct inoculation from the rectal swabAfter enrichment in 0.5 μg/ml ertapenem containing BHIKPCNDMVIMIMPOXA-48-likeAcOXAMCRGES
Negative (60)Negative (60)---------
E. coli + K. pneumoniae (2)Not performedKPC-223–24-------
NegativeK. pneumoniaeKPC-325-------
Negative (GeneXpert result :VIM)NegativecVIM-type--33-----
E. coliNot performedNDM-5-31------
K. pneumoniae (1), A. baumannii (1)Not performedNDM-1-32------
K. pneumoniae + A. baumannii (3)Not performedNDM- 1 + OXA-23-23–29---25–27--
NegativeA. baumanniiNDM-1-33---- (38)d--
NegativeA. baumanniiNDM-1-- (39)------
NegativeA. baumanniiNDM-1--------
A. baumannii (2)Not performedOXA-23-----15–22--
C. freundii (1), E. cloacae + K. varicola (2), E. cloacae (4)Not performedOXA-48----18–25---
E. aerogenesNot performedOXA-48----34---
E. coli (6), E. coli + C. freundii (1)Not performedOXA-48----19–29---
K. pneumoniae (3), E. coli + K. pneumoniae (5)Not performedOXA-48----19–30---
E. cloacaeeNot performedOXA-48 + OXA-23----2327--
K. oxytocafNot performedOXA-48----- (37)---
a
n = 100. Sensitivity, 92.5% (95% CI, 78.5%–98.0%); specificity, 100% (95% CI, 92.5%–100%). Positive predictive value, 100% (95% CI, 88.3%–100%). Negative predictive value, 95.2% (95% CI, 85.8%–98.8%). Dark gray boxes represent discrepant results. -, negative result.
b
-, no amplification.
c
No growth was detected on ChromID Carba SMART after plating 100 μl of an overnight culture of the ESwab in brain heart infusion supplemented with 0.5 μg/ml ertapenem.
d
The negativity cutoff value being fixed at CT ≥ 35 by the manufacturer, this sample is counted as negative.
e
Although no culture of A. baumannii was obtained with this sample, an in-house PCR confirmed the presence of the blaOXA-23 gene.
f
Only 2 CFU were obtained on ChromID Carba Smart.

ACKNOWLEDGMENTS

This work was supported by the Assistance Publique–Hôpitaux de Paris (AP-HP), the University Paris-Sud, 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).
We declare that we have conflicts of interest.

REFERENCES

1.
Logan LK, Weinstein RA. 2017. The epidemiology of carbapenem-resistant Enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis 215:S28.
2.
Gniadek TJ, Carroll KC, Simner PJ. 2016. Carbapenem-resistant non-glucose-fermenting gram-negative bacilli: the missing piece to the puzzle. J Clin Microbiol 54:1700–1710.
3.
Sun J, Zhang H, Liu Y-H, Feng Y. 2018. Towards understanding MCR-like colistin resistance. Trends Microbiol 26:794–808.
4.
Beyrouthy R, Robin F, Lessene A, Lacombat I, Dortet L, Naas T, Ponties V, Bonnet R. 2017. MCR-1 and OXA-48 in vivo acquisition in KPC-producing Escherichia coli after colistin treatment. Antimicrob Agents Chemother 61:e02540-16.
5.
Oueslati S, Girlich D, Dortet L, Naas T. 2018. Evaluation of the Amplidiag CarbaR+VRE kit for the accurate detection of carbapenemase-producing bacteria. J Clin Microbiol 56:01092-17.
6.
Girlich D, Naas T, Dortet L. 2018. Comparison of the Superpolymyxin™ and CHROMID Colistin R screening media for the detection of colistin-resistant Enterobacteriaceae from spiked rectal swabs. Antimicrob Agents Chemother AAC.01618-18.
7.
Bonnin RA, Nordmann P, Poirel L. 2013. Screening and deciphering antibiotic resistance in Acinetobacter baumannii: a state of the art. Expert Rev Anti Infect Ther 11:571–583.
8.
Poirel L, Figueiredo S, Cattoir V, Carattoli A, Nordmann P. 2008. Acinetobacter radioresistens as a silent source of carbapenem resistance for Acinetobacter spp. Antimicrob Agents Chemother 52:1252–1256.
9.
Plésiat P, Cattoir V, Bonnet R, Naas T. 2016. 2016 Activity report. National Reference Center for Antibiotic Resistance. http://www.cnr-resistance-antibiotiques.fr/ressources/pages/Rapport_CNR_2016.pdf.

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cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 57Number 3March 2019
eLocator: e01800-18
Editor: Karen C. Carroll, Johns Hopkins University School of Medicine
PubMed: 30541941

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Published online: 27 February 2019

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Delphine Girlich
EA7361 “Structure, dynamic, function and expression of broad spectrum β-lactamases,” Université Paris-Sud, Université Paris-Saclay, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France
Evolution and Ecology of Resistance to Antibiotics Unit, Institut Pasteur, APHP, Université Paris-Sud, Paris, France
Sandrine Bernabeu
EA7361 “Structure, dynamic, function and expression of broad spectrum β-lactamases,” Université Paris-Sud, Université Paris-Saclay, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France
Evolution and Ecology of Resistance to Antibiotics Unit, Institut Pasteur, APHP, Université Paris-Sud, Paris, France
Bacteriology-Hygiene Unit, Assistance Publique–Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France
Associated French National Reference Center for Antibiotic Resistance, Le Kremlin-Bicêtre, France
Victor Grosperrin
EA7361 “Structure, dynamic, function and expression of broad spectrum β-lactamases,” Université Paris-Sud, Université Paris-Saclay, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France
Isabelle Langlois
Bacteriology-Hygiene Unit, Assistance Publique–Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France
Christine Begasse
Bacteriology-Hygiene Unit, Assistance Publique–Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France
Nicolas Arangia
Bacteriology-Hygiene Unit, Assistance Publique–Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France
Elodie Creton
Associated French National Reference Center for Antibiotic Resistance, Le Kremlin-Bicêtre, France
Garance Cotellon
Associated French National Reference Center for Antibiotic Resistance, Le Kremlin-Bicêtre, France
Aimie Sauvadet
Associated French National Reference Center for Antibiotic Resistance, Le Kremlin-Bicêtre, France
Laurent Dortet
EA7361 “Structure, dynamic, function and expression of broad spectrum β-lactamases,” Université Paris-Sud, Université Paris-Saclay, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France
Evolution and Ecology of Resistance to Antibiotics Unit, Institut Pasteur, APHP, Université Paris-Sud, Paris, France
Bacteriology-Hygiene Unit, Assistance Publique–Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France
Associated French National Reference Center for Antibiotic Resistance, Le Kremlin-Bicêtre, France
EA7361 “Structure, dynamic, function and expression of broad spectrum β-lactamases,” Université Paris-Sud, Université Paris-Saclay, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France
Evolution and Ecology of Resistance to Antibiotics Unit, Institut Pasteur, APHP, Université Paris-Sud, Paris, France
Bacteriology-Hygiene Unit, Assistance Publique–Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France
Associated French National Reference Center for Antibiotic Resistance, Le Kremlin-Bicêtre, France

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Karen C. Carroll
Editor
Johns Hopkins University School of Medicine

Notes

Address correspondence to Thierry Naas, [email protected].

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