Clinical Microbiology and Infection xxx (xxxx) xxx

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Clinical Microbiology and Infection

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Original article

Comparison of five methods for detection of carbapenemases in

Enterobacterales with proposal of a new algorithm
€ ttig 3, A. Saleh 1, Y. Stelzer 1,
L. Lucena Baeza 1, N. Pfennigwerth 2, C. Greissl 1, S. Go
S.G. Gatermann 2, A. Hamprecht 1, 4, *
1)
Institute for Medical Microbiology, Immunology and Hygiene, University Hospital of Cologne, Cologne, Germany
2)
Department of Medical Microbiology, Ruhr University Bochum, Bochum, Germany
3)
Institute of Medical Microbiology and Infection Control, Hospital of Johann Wolfgang Goethe-University, Frankfurt am Main, Germany
4)
DZIF (German Centre for Infection Research), Partner Site Bonn-Cologne, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: The aim of this study was to evaluate the performance of five different carbapenemase tests
Received 14 December 2018 and to develop an algorithm which will permit the detection of most common and rare carbapenemases
Received in revised form in routine microbiology laboratories.
1 March 2019
Methods: The immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric
Accepted 2 March 2019
Available online xxx
b-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc
(zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly charac-
Editor: F. Allerberger terized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n ¼ 48), OXA-48-
like (n ¼ 40), NDM (n ¼ 29), KPC (n ¼ 13), IMI (n ¼ 9), IMP (n ¼ 9), OXA-58 (n ¼ 2), and GES (n ¼ 2).
Keywords: Results: The overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2%
b-CARBA (CI 77.6e89.2%)/100% (CI 91.8e100%) for RESIST-4, 88.2% (CI 82.1e92.4%)/100% (CI 91.8e100%) for
CARBA-5 CARBA-5, 88.2% (CI 82.1e92.4%)/100% (CI 91.8e100%) for Xpert Carba-R, 73.7% (CI 66.2e80.0%)/100% (CI
Carbapenem-inactivation method 93.4e99.0%) for b-CARBA, and 97.4% (CI 87.9e99.6%)/97.7% (CI 87.9e99.6%) for zCIM. The four common
GeneXpert
carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with 97.6% sensitivity by all tests
Immunochromato graphic assay
except for b-CARBA (76.6% (CI 68.4e83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9%
IMP
KPC (CI 62.3e98.4%)). Based on these results a new algorithm was developed, consisting of an immuno-
NDM chromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carba-
OXA-48 penemases assessed.
VIM Conclusions: Except for b-CARBA, all methods showed excellent sensitivity/specificity for the detection of
the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is
rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equip-
ment is required. L. Lucena Baeza, Clin Microbiol Infect 2019;▪:1
© 2019 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and
Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction permeability or by the production of carbapenemases (carbapen-

The worldwide emergence of antibiotic resistance severely
limits therapeutic options and is a significant global public health
threat [1]. Resistance to carbapenems in Enterobacterales can be
caused by hyperproduction of AmpC b-lactamases or extended-
spectrum b-lactamases (ESBLs) combined with altered membrane
* Corresponding author: A. Hamprecht, University of Cologne, Institute for
Medical Microbiology Immunology and Hygiene, Cologne, Germany.
E-mail address: axel.hamprecht@uk-koeln.de (A. Hamprecht).
emase-producing Enterobacterales (CPE)). To contain the further
dissemination of CPE, the rapid and accurate identification of car-
bapenemases is of paramount importance for both epidemiological
and infection control purposes [2]. Additionally, rapid identification
of some carbapenemases can help to guide therapy, as most isolates
producing OXA-48-like or Klebsiella pneumoniae carbapenemases
(KPC) are susceptible to ceftazidimeeavibactam.
Although a number of phenotypic and genotypic diagnostic
methods for CPE detection have been developed, the performance
of these tests varies considerably, depending on the enzyme and
species. Colorimetric tests, such as the RAPIDEC CARBA NP

https://doi.org/10.1016/j.cmi.2019.03.003
1198-743X/© 2019 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Baeza LL et al., Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a
new algorithm, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.03.003
1.e2 L.L. Baeza et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
(bioMerieux, Nürtingen, Germany) or the b-CARBA (BioRad,
Marnes-la-Coquette, France), show variable sensitivity for b-lacta-
mases with low carbapenemase activity [3]. The carbapenem
inactivation method (CIM) [4] or its modified version (mCIM) [5,6]
require an extra overnight culture for a definitive result and hence
delay the time-to-result. These phenotypic assays detect carbape-
nem hydrolysis but cannot identify the respective carbapenemase.
Immunochromatographic tests (ICT), such as the recently devel-
oped RESIST-4 O.K.N.V. (Coris BioConcept, Gembloux, Belgium) or
the CARBA-5 (CARBA-5, NG biotech, Guipry, France), represent the
fastest methods available, demonstrating high sensitivity and
specificity in several studies [7e9]. PCR is considered as the refer-
ence standard for carbapenemase detection, but it requires addi-
tional equipment, skilled staff and is not available in many
laboratories, especially beyond core working hours. Additionally,
only targeted genes can be detected, with new enzyme variants
possibly being missed.
The goal of this study was to evaluate the performance of five
carbapenemase detection methods in Enterobacterales which are
suitable for routine microbiology laboratories. Two ICTs, a colori-
metric test, a modified CIM test (zCIM), and the automated qPCR-
based GeneXpert Carba-R assay (Cepheid, Frankfurt, Germany)
were compared. Based on our results, an algorithm for improved
detection of carbapenemases in the routine microbiology labora-
tory was developed.
Materials and methods
Bacterial isolates
A total of 189 clinical Enterobacterales isolates, all molecularly
characterized by PCR and sequencing of carbapenemase genes,
were employed to challenge the assays. All clinical isolates were
obtained from hospitalized patients in Germany, either from pre-
vious studies [10e15] or from the German National Reference
Center for multidrug-resistant Gram-negative bacteria at the Uni-
versity of Bochum. The collection was composed of 43
carbapenemase-negative isolates and 146 CPE producing a total of
152 carbapenemases, including isolates belonging to Ambler clas-
ses A (n ¼ 22), B (n ¼ 80), D (n ¼ 38), and six isolates producing two
carbapenemases (Table 1). Out of all carbapenemase-negative
strains (n ¼ 43), 21 (48.8%) were non-susceptible to at least erta-
penem (MIC 1 mg/L), mostly by a combination of ESBL (n ¼ 27)
and/or AmpC (n ¼ 12) and porin loss. For further characterization of
isolates please see supplementary material.
For the comparison of the assays, a suspension equivalent to a
0.5 McFarland turbidity standard was inoculated on
MuellereHinton agar plates (MHA; Oxoid, Wesel, Germany) and
incubated overnight at 37 C. All five methods were performed on
the same day using the same inoculum. Except for the Xpert Carba-
R (automated interpretation), all other tests were read by a second
person who was blinded to the molecular characterization. All tests
were performed according to the manufacturer's recommendations
unless stated otherwise.
Carbapenem inactivation method
For assessment of CIM and its modified versions, a series of
different conditions were tested on a preliminary collection of 46
carbapenemase-positive and 11 carbapenemase-negative isolates
(see Table S1). A full 10-mL inoculation loop of bacteria was sus-
pended in 400 mL of Tryptic Soy Broth (TSB; BD, Heidelberg, Ger-
many) or distilled water with or without ZnSO4 (0.3 mM final
concentration), and a 10-mg meropenem disc (Oxoid). After a 2-hr
incubation at 37 C, the disc was placed on a MHA plate
Please cite this article as: Baeza LL et al., Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a
new algorithm, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.03.003
previously inoculated with the susceptible Escherichia coli indicator
strain ATCC 25922 (0.5 McFarland). After an 18-hr incubation at
37 C, the inhibition zone was measured and the presence of
microcolonies was recorded. If available, cut-offs published previ-
ously were used [4e6]; additionally new cut-offs were calculated
based on the frequencies of CIM inhibition zone measurements (see
Figs S3eS6). Among all conditions tested, TSB þ ZnSO4 (¼ zCIM)
showed the overall best performance among the preliminary bac-
terial collection (sensitivity 97.8%, specificity 100%) using the cutoff
20 mm/>20 mm for positive/negative (Fig. S6 and Table S7). It was
therefore chosen for the following evaluation of 189 isolates.
Cepheid Xpert Carba-R
A full 10-mL inoculation loop with a bacterial suspension
equivalent to 0.5 McFarland standard was added into the sample
reagent. After homogenization by vortexing for 10 s, 1.7 mL was
subsequently transferred into the Xpert Carba-R cartridge (version
2.0) and run on the GeneXpert platform (Cepheid).
b-CARBA test
The b-CARBA test (BioRad) was performed as follows: for every
sample, 40 mL of reagent 1 was mixed with 40 mL of reagent 2. A full
1-mL inoculation loop was harvested from MHA, mixed with the
reagents, vortexed, and incubated at 37 C for 30 min before results
were read. The protocol used here deviated from the manufac-
turer's recommendations by the use of MHA instead of other media
(e.g. Columbia blood agar (CBA)).
CARBA-5
Five drops of extraction buffer were mixed with a full 1-mL
inoculation loop of bacteria harvested from MHA and 100 mL of this
suspension was transferred into the CARBA-5 cassette (NG
biotech); results were read after 15 min of incubation at room
temperature.
RESIST-4 O.K.N.V
The test was performed as previously published [16]. Briefly, 12
drops of LY-A buffer were mixed with a full 1-mL inoculation loop of
bacteria, which has previously been demonstrated to be the
optimal amount for analysis [10]. Three drops of diluted sample was
added into each well of the two cassettes of RESIST-4 O.K.N.V.
(Coris). Results were read after 15 min of incubation at room
temperature.
Statistics
The sensitivity and specificity was calculated and comparison
with molecular characterization which served as the reference
standard. Additionally, the 95% confidence intervals (CIs) and the
Youden index were calculated.
Results
Comparison of the performances of carbapenemase-detection
methods in Enterobacterales
The overall sensitivity of the five tests was 73.7% (CI 66.2e80.0%)
for b-CARBA, 84.2% (CI 77.6e89.2%) for RESIST-4, 88.2% (CI
82.1e92.4%) for CARBA-5, 88.2% (CI 82.1e92.4%) for Xpert Carba-R,
and 97.4% (CI 93.4e99.0%) for zCIM (Table 2). When considering
only the four most common carbapenemases (NDM, KPC, OXA-48,
 L.L. Baeza et al. / Clinical Microbiology and Infection xxx (xxxx) xxx 1.e3

Table 1
Overview of the final bacterial isolate collection used for the evaluation of the five carbapenemase detection methods

K. pneumoniae E. coli E. cloacae C. freundii E. aerogenes S. marcescens P. mirabilis Others* Subtotal

Carbapenemase-positive 47 32 32 19 2 5 3 6 146
Ambler class A 9 9 2 2 22
GES 1 1 2
GES-2 1 1
GES-25 1 1
IMI 9 9
IMI-1 1 1
IMI-2 1 1
IMI-3 1 1
IMI-4 1 1
IMI-9 1 1
IMI-10 1 1
IMI-12 1 1
IMI-14 1 1
IMI-16 1 1
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 2 38
OXA
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
OXA-58 2 2
Isolates with 2 carbapenemases 3 3 6
OXA-232þNDM-1 1 1
KPC-2þVIM-1 2 2
OXA-181þNDM-5 1 1
OXA-48þNDM-1 1 1
OXA-48þVIM-1-like 1 1
Carbapenemase-negative 10 20 6 1 4 1 1 43
Total number 57 52 38 20 6 5 4 7 189
*
VIM-1 Escherichia hermannii (n ¼ 1), GES-2 Klebsiella aerogenes (n ¼ 1), Klebsiella oxytoca (VIM-2, n ¼ 1 and VIM-4, n ¼ 1), KPC-2 Citrobacter braakii (n ¼ 1), NDM-1
Raoultella ornithinolytica (n ¼ 1) and carbapenemase-negative Klebsiella oxytoca (n ¼ 1). The main carbapenemase groups are marked in bold.

Please cite this article as: Baeza LL et al., Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a
new algorithm, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.03.003
1.e4 L.L. Baeza et al. / Clinical Microbiology and Infection xxx (xxxx) xxx

VIM), the sensitivity was 77.7% (CI 69.8e84.0%) for b-CARBA, 98.5%

Spec % (CI) Youden index

(CI 94.6e99.6%) for RESIST-4, 99.2% (CI 95.8e99.9%) for CARBA-5,
100% (CI 97.1e100%) for Xpert Carba-R, and 97.7% (CI 93.4e99.2%)
for zCIM (Table S8). KPC and OXA-48-like were detected 100% (CI

0.95
93.2e100%) by all tests, while NDM and VIM were more frequently
missed, with the lowest sensitivity being recorded for b-CARBA

(87.9e99.6)
(41.4%, CI 25.5e59.3%, and 75.0%, CI 61.2e85.1%, respectively). Of
note, in isolates co-producing NDM and OXA-48-like the carba-

97.4 (93.4e99.0) 97.7

penemase NDM was not detected by CARBA-5 in one out of three

96.5 (90.2e98.8)

93.8 (83.2e97.9)
95.8 (79.8-99.3)

100 (77.2e100)

100 (70.1e100)

100 (88.3e100)

100 (70.1e100)
100 (91.6e100)

100 (91.2e100)

100 (34.2e100)
and RESIST-4 in two out of three isolates.

50 (9.5e90.6)
b-CARBA and zCIM were the only assays which were positive in

Spec % (CI) Youden Sens % (CI)

all IMP-producing isolates (sensitivity 100%, CI 70.1e100%), fol-

zCIM
lowed by CARBA-5 (sensitivity 55.6%, CI 26.7e81.1%), detecting
IMP-1/-4/-8/-22 and Xpert Carba R (sensitivity 44.4%, CI
18.9e73.3%), detecting IMP-1/-4/-28), see Table S9. All IMI vari-

index

0.73
ants and one GES-25 were only detected by zCIM, as they are not
targeted by the ICTs or Xpert Carba-R. OXA-58 was only detected

(91.8e100)
using zCIM and b-CARBA. All assays showed 100% (CI 91.8e100%)
specificity except for the zCIM (97.7%, CI 87.9e99.6%). Only one

73.7 (66.2e80.0) 100

AmpC producing Enterobacter cloacae isolate with decreased

54.2 (35.1e72.1)

66.3 (55.8e75.4)

41.4 (25.5e59.3)
75.0 (61.2e85.1)
permeability gave rise to a false-positive result for zCIM, while

100 (77.2e100)

100 (70.1e100)
100 (91.6e100)

100 (91.2e100)

100 (34.2e100)
other tests did not produce any false-positive results. The Youden

Spec % (CI) Youden Sens % (CI)

0 (0e65.8)
0 (0e29.9)
index ranged from 0.73 for b-CARBA to 0.95 for zCIM (Table 2).

b-CARBA
A comparison describing the main features of each test is
shown in Table 3.

index

0.88
Development of an algorithm for the detection of carbapenemases
in the clinical laboratory.

(91.8e100)
Since none of the assays detected all carbapenemases an al-
88.2 (82.1e92.4) 100
gorithm was developed combining two different methods
54.2 (35.1e72.1)

94.2 (87.1e97.5)

44.4 (18.9e73.3)
95.2 (84.2e98.7)
(Table S10). The overall highest sensitivity of 99.3% (CI

100 (77.2e100)

100 (88.3e100)
100 (92.6e100)

100 (91.2e100)
Xpert Carba R

96.2e99.9%)/specificity 97.7% (CI 87.9e99.6%) can be achieved

0 (0.0e29.9)
Spec % (CI) Youden Sens % (CI)

0 (0e65.8)

0 (0e65.8)
with the combination of zCIM with either CARBA-5, Carba-R or
RESIST-4. If CARBA-5 or RESIST-4 are used as a first assay, the four
most common carbapenemases are detected within 15 min
(Fig. 1). If positive, no further testing is necessary. In case of a
index

0.88

negative result, zCIM is performed as a second test for phenotypic

detection of rare carbapenemases. If zCIM is also negative, a
(91.8e100)

carbapenemase is highly unlikely. If zCIM results positive, further

work-up is necessary, e.g. by referring to a reference centre, by
88.2 (82.1e92.4) 100

additional PCRs for rare carbapenemases or by whole-genome

sequencing if available.
54.2 (35.1e72.1)

94.2 (87.1e97.5)

96.6 (82.8e99.4)

55.6 (26.7e81.1)
95.2 (84.2e98.7)
100 (77.2e100)

100 (92.6e100)

100 (91.2e100)
Performance of the five assays for detection of carbapenemase production

0 (0.0e65.8)
Spec % (CI) Youden Sens % (CI)

0 (0e65.8)
0 (0e29.9)

Discussion
CARBA-5

In this study, we assessed the performance of five phenotypic

and genotypic methods for carbapenemase detection using a large
index

Sens, sensitivity; Spec, specificity; CI, confidence interval.

collection of clinical Enterobacterales isolates and evaluated their

0.84

applicability in the clinical setting.

(91.8e100)

Several studies assessing the immunochromatographic

RESIST-3 O.K.N. have been published, showing high sensitivity and
84.2 (77.6e89.2) 100

specificity results [10,15,17]. Only two studies analysed the per-

RESIST-4 O.K.N.V.

formance of RESIST-4 O.K.N.V., demonstrating 100% sensitivity for

54.2 (35.1e72.1)

87.2 (78.5e92.7)

93.1 (78.0e98.1)

95.2 (84.2e98.7)
100 (77.2e100)

100 (92.6e100)

100 (91.2e100)

KPC, OXA, and VIM variants [9,16]. For NDM a sensitivity of 83.3%
0 (0.0e65.8)
Sens % (CI)

0 (0e65.8)
0 (0e29.9)

0 (0e29.9)

was recorded in one study as a result of two false-negatives (one

Proteus mirabilis and one Providencia stuartii) [9]. In the second
study, the sensitivity for NDM was 96.6% when tested from MHA,
but increased to 100% when tested from CBA, zinc-supplemented
All carbapenemases

OXA-58 (n ¼ 2)

MHA or from the inhibition zone of an ertapenem disc [16]. The

NDM (n ¼ 29)
VIM (n ¼ 48)
KPC (n ¼ 13)

Ambler class D
Ambler class A

Ambler class B

OXA-48-like

false-negative isolate from MHA was also an isolate of P. mirabilis.

IMP (n ¼ 9)
GES (n ¼ 2)
IMI (n ¼ 9)

(n ¼ 40)
(n ¼ 152)

(n ¼ 24)

(n ¼ 86)

(n ¼ 42)

In the present study, the only NDM-producing P. mirabilis tested

positive, but two isolates co-producing NDM were false negatives.
Table 2

CARBA-5 detected all KPC, OXA, VIM, and NDM (except for one
K. pneumoniae co-producing OXA-232/NDM-1), which is in

Please cite this article as: Baeza LL et al., Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a
new algorithm, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.03.003
new algorithm, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.03.003
Please cite this article as: Baeza LL et al., Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a

Table 3
Comparison of main features of each carbapenemase detection method

RESIST-4 O.K.N.V. CARBA-5 b-CARBA zCIM XpertCarba-R

Special equipment All supplied by manufacturer All supplied by manufacturer All supplied by manufacturer Conventional laboratory equipment GeneXpert instrument
necessary
Storing conditions Room temperature Room temperature 4 C 4 C Room temperature
Time for
Preparation 5 min 5 min 5 min 10 min 10 min

L.L. Baeza et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
Incubation/ 15 min 15 min 30 min 2 h plus overnight culture 1 hour
reaction
Reading/ 1 min 1 min 1 min 5 min 1 min
Interpretation
Definitive result 21 min 21 min 36 min 20 h 15 min 1 h11 min
Price per test* ~15V ~15V ~5V ~1V ~30V
Test principle Immunochromatographic test Immunochromatographic test Colorimetric test Carbapenem hydrolysis assay Multiplex-PCR
(activity test)
Result OXA-48 ,KPC, NDM, VIM OXA-48, KPC, NDM, VIM, some Carbapenemase positive/negative Carbapenemase positive/negative OXA-48, KPC, NDM,
IMP variants VIM, some IMP variants
Sample type Bacterial colonies, some clinical Bacterial colonies Bacterial colonies, some clinical Bacterial colonies Bacterial colonies, some
samples (e.g.urine or samples (blood cultures [28]) clinical samples [29]
blood cultures [15])
Interpretation Straightforward for most isolates; Straightforward for most Subjective (colour change) Subjective if microcolonies present Automatic interpretation
faint bands can occur for some isolates;
NDM/VIM isolates faint bands can occur for some
NDM/VIM isolates
Strengths Fast, simple moderate costs Fast, simple moderate costs Fast, simple differentiation Simple, inexpensive differentiation High reproducibility
differentiation of the 4 most differentiation of the 5 most CPE/non-CPE only CPE/non-CPE relatively fast
common carbapenemases common carbapenemases detection of some rare detection of rare/new carbapenemases differentiation of the 5most
carbapenemase variants common carbapenemases
(e.g. OXA-58) possible
Limitations False-negatives with weak MBLs False-negatives with weak or low-level carbapenemase False-negatives with False-positives in some isolates with Expensive
when harvested from producers, especially withMBLs weak or low-level AmpC and porin loss Additional equipment
MHA in absence of antibiotic carbapenemase producers, False-negatives with weak carbapenemases required
pressure [10,16] especially with Long time-to-result
MBLs and IMI/GES
Reference [9] [7] [3] This study [18]
*
Subject to variation depending on country/distributor.

1.e5
1.e6 L.L. Baeza et al. / Clinical Microbiology and Infection xxx (xxxx) xxx

agreement with previous results [7,8]. This study demonstrated the
additional detection of NDM-8, VIM-5/-26/-27/-31/-39/-46/-51/-
52/-54/-56/-58/-59, OXA-370, and IMP-22 (Table S9). IMP-13/-14/-
28/-50 were not detected, with IMP-13/-14 proving also negative as
published elsewhere [8].
Our findings with Xpert Carba-R are in agreement with the
manufacturer's specification and with results from several studies,
which demonstrated that only IMP variants belonging to the IMP-1
family can be detected [18e20]. Xpert Carba-R was the only assay
which detected all carbapenemases in double-carbapenemase
producers.
The b-CARBA test proved to be the least sensitive of all methods
evaluated here. Several studies showed a wide range of sensitivity,
ranging from 64.9% to 97.3% [3,21e24]. The performance depends
on the carbapenemase but is also considerably influenced by the
inoculum, incubation temperature, and time [3,21]. The overall
sensitivity found in the present study was 73.7%, which is mainly
the result of the poor detection of MBLs. This could be related to
culture on MHA, which is a medium not recommended for this test.
When retested from CBA or MHA supplemented with 50 mg/L zinc
sulphate, sensitivity improved to 95.9% (CI 91.3e98.1%) and 96.6%
(CI 92.2e98.5%), respectively. A better performance from CBA or
zinc supplemented agars, which increase the detection rate of MBL
producers due to higher enzymatic activity in the presence of zinc,
has also been shown for other tests, e.g. ICTs [10,15,16].
The widest spectrum of carbapenemases was detected by zCIM,
including rare variants. A positive zCIM test indicates hydrolytic
activity and is therefore able to detect also carbapenemases not
targeted by the ICTs or Xpert Carba-R. While CIM and mCIM have
been shown to be highly sensitive and specific [4,25,26], among our
isolates MBLs were only partly detected with the original CIM test.
For that reason, we developed zCIM, which showed improved
sensitivity (97.8%) compared with the original CIM (69.6%; Table S7)
and has a shorter incubation time (2 hr) than mCIM (4 hr).
Inhibitor-based combination disc tests (e.g. with EDTA and boronic
acid) are recommended by EUCAST [27] for carbapenemase
detection and could be an easy-to-perform alternative to zCIM
(please see supplementary material). Among the 22 isolates pro-
ducing IMI, GES, IMP, and OXA-58 which are currently not or only
partly detected by CARBA-5, RESIST-4, or Xpert Carba-R, the
sensitivity of inhibitor tests was 13/22 (59.1%, CI 38.7e76.7%).
When testing all 189 isolates with inhibitors, sensitivity was higher
(84.9%, CI 78.2e89.8%), but specificity was moderate (76.7%, CI
62.3e86.9%).
For optimized carbapenemase detection, the new algorithm
incorporating an ICT þ zCIM (Fig. 1) has the advantage of being
rapid, affordable, and simple. With such a combination, the four
most common carbapenemases can be identified within
15e20 min, with the less frequent ones being detected within a day.
Compared with a single test (sensitivity 73.7e97.4%), the combi-
nation of either CARBA-5 or RESIST-4 with zCIM improves sensi-
tivity to 99.3%.
This study has several limitations. We used MHA since addi-
tional tests are mostly done from this agar, e.g. after carbapenem
non-susceptibility has been detected by a disc diffusion test.
Therefore, the use of MHA is likely to produce the result most close
to real life in the routine laboratory. Additionally, this was an
analysis based on a collection containing many rare carbapene-
mases in order to challenge the tests. The performance of the assays
is likely to be better in the routine laboratory, where the four main
carbapenemases mostly occur. When taking into account only OXA-
48-like, KPC, NDM, and VIM, the overall sensitivity was higher in all

Fig. 1. Algorithm for the detection of carbapenemases in the routine laboratory.*OXA-48-like, KPC, VIM, NDM, (IMP) **Multiplex PCR targeting rare carbapenemases (e.g.IMI, GES
etc.), whole genome sequencing, other tests.

Please cite this article as: Baeza LL et al., Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a
new algorithm, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.03.003
 L.L. Baeza et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
assays (98.5% for RESIST-4, 99.2% for CARBA-5, 100% for Xpert
Carba-R, 77.7% for b-CARBA, and 97.7% for zCIM) (Table S8).
To the best of our knowledge this is the first study to compare
the two ICTs RESIST-4 and CARBA-5 and also the first to compare
ICTs to the Xpert Carba-R or the b-CARBA. A large number of
molecularly characterized isolates were analysed, comprising 53
different carbapenemases in 12 species. All assays were evaluated
in a blinded fashion to exclude reading bias. Furthermore, an easy
algorithm for improved detection was developed which will permit
the detection of a wide range of carbapenemases in the routine
microbiology laboratories without the need for molecular methods
or additional equipment.
In conclusion, most evaluated assays showed good sensitivity
and specificity, at least for the common carbapenemase families
OXA-48-like, KPC, VIM, and NDM and are suitable for the routine
microbiology laboratory, thereby decreasing the time to detection
and the need to refer these isolates to reference laboratories.
However, the detection of rare carbapenemases (e.g. IMI, GES, IMP,
or OXA-58) is still problematic with most of the commercially
available tests. The combination of two tests, e.g. CARBA-5 þ zCIM,
will enable most laboratories to detect these rare variants at low
costs. This could help to optimize patient treatment and to limit the
further spread of CPE.
Transparency declaration
A.H. has received lecture honoraria from OXOID, Becton Dick-
inson and Bruker Daltonics, outside the submitted work. S.G.G.
received lecture honoraria from Beckman Coulter and bioMe rieux,
outside the submitted work. All other authors declare no conflict of
interest. This study was funded by grants from the Faculty of
Medicine, University of Cologne and supported by the Robert Koch
Institute with funds provided by the German Ministry of Health
(grant no. 1369-402). AH was supported by the DZIF (German
Centre for Infection Research).
Acknowledgements
RESIST-4 tests were provided by Coris BioConcept and CARBA-5
tests by Virotech Diagnostics Germany/NG Biotech free of charge.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.cmi.2019.03.003.
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