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Vol. 41, No. 2

January 15, 2019
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I n Th is Issu e
11 Phenotypic Methods
for Detection of
Carbapenemase Production
in Carbapenem-Resistant
Organisms: What Method
Should Your Laboratory
Choose?
Phenotypic Methods for Detection of Carbapenemase
Production in Carbapenem-Resistant Organisms: What
Method Should Your Laboratory Choose?
Meklit Workneh, M.D., M.P.H 1., Rebecca Yee, Ph.D.,2 and Patricia J. Simner, Ph.D.,1 1Johns Hopkins
University School of Medicine, Department of Pathology, Division of Medical Microbiology, 2Johns Hopkins
Bloomberg School of Public Health, Baltimore, Maryland
Abstract
Antimicrobial resistance is a rising problem among Gram-negative organisms, and resistance to carba­
penems is a special concern and an urgent public health threat. Rapid detection of carbapenem resis-
tance, and carbapenemase production specifically, is becoming increasingly important for guiding
infection control strategies and for therapeutic management of patients. Several types of phenotypic
tests for the detection of carbapenemase production continue to be developed and include culture/
growth-based methods (carbapenem inactivation method [CIM]/modified CIM [mCIM]), colorimetric
assays (Carba NP and derivatives), matrix-assisted laser desorption ionization–time of flight mass spec-
trometry (MALDI-TOF MS)-based tests, and lateral-flow assays. Here, we describe the tests currently
available, their performance characteristics, and how to select and verify/validate a test for the specific
needs of a laboratory.

Introduction are β-lactamase enzymes capable of cleaving the

Corresponding author:
Patricia J. Simner, Ph.D.,
D(ABMM), Division of Medical
Microbiology, Department of
Pathology, Meyer B1-193,
600 N. Wolfe St., Baltimore,
MD 21287-7093. Tel.:
410-955-5077. E-mail:
psimner1@jhmi.edu
0196-4399/©2019 Elsevier Inc.
All rights reserved
Multidrug resistance is a rising problem among
Gram-negative organisms. Carbapenems are
broad-spectrum β-lactam antibiotics used as a
last line of defense for multidrug-resistant organ-
isms (MDROs). Increasing resistance to these
last-line agents is now being seen, and there is
growing urgency to rapidly detect and respond
to MDROs that are also carbapenem resistant.
Rapid detection of carbapenem resistance and
delination of mechanism of resistance is impor-
tant from a clinical treatment standpoint, but is
also necessary for implementation of appropriate
infection control strategies, as well as for out-
break detection and investigation [1,2].
Mechanisms of Carbapenem Resistance
A number of different mechanisms, which can
be classified as carbapenemase producing (CP)
or non-carbapenemase producing (non-CP),
can mediate carbapenem resistance among
Gram-negative organisms. Carbapenemases
amide bond of most β-lactam rings, including
the carbapenems, rendering them inactive [3-5].
The non-CP carbapenem-resistant organisms
(CRO) produce other mechanisms of resistance,
including porin mutations or efflux pumps, or
a combination of these, with the production
of extended-spectrum β-lactamase (ESBL) or
AmpC β-lactamase, depending on the specific
Gram-negative organism [3-5]. All CRO are of
concern, as they are likely to be multidrug resis-
tant (MDR). However, the CP CRO are most
concerning and are more aggressively targeted
by antibiotic stewardship and infection control
programs, as they are thought to be the primary
mechanism responsible for driving the spread of
carbapenem resistance among Gram-negative
organisms globally [1]. These clinically impor-
tant pathogens are considered a triple threat due
to their increasing prevalence, their MDR pro-
file, and their ease of transmission from species
to species and even among different genera of

Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier 11

Gram-negative bacteria through transmissible genetic elements
(i.e., transposons, insertion sequences, and plasmids). Moreover,
the transmissible genetic elements carry multiple additional antibi-
otic resistance genes, resulting in MDR or extensively drug-resis-
tant profiles, greatly limiting therapeutic options. Most concerning
of all is the associated mortality of up to 60% in patients infected
with CP CRO [6].
Classification of Carbapenemase Enzymes
The carbapenemases are grouped into three classes of β-lactamases:
Ambler classes A, B, and D. Ambler class A includes Klebsiella pneu-
moniae carbapenemase (KPC), Serratia marcescens enzyme (SME),
imipenemase (IMI), non-metallocarbapenemase-A (NMC), and
Guiana extended-spectrum (GES) enzymes. Geographic varia-
tion can be seen in the prevalence of the different enzymes, with
the KPC enzyme being the most common type of carbapen-
emase in the United States [7,8]. Ambler class B includes New-
Delhi metallo-beta-lactamase (NDM), Verona integron-borne
metallo-beta-lactamase (VIM), and imipenemase (IMP) types,
which are also known as the metallo-β-lactamases (MBLs), as
they require divalent cations, usually zinc, as metal cofactors for
enzyme activity. Ambler Class D includes only the OXA enzymes
[7,8]. Different enzymes have predilections for different organism
groups, with KPC enzymes being more common in the Entero-
bacteriaceae, for example [7,8], while certain OXA carbapenemases
(OXA-23, OXA-24/40, OXA-58, and OXA-143) have been his-
torically found more commonly in Acinetobacter spp. [9], with the
OXA-48 group now emerging rapidly among the Enterobacteriaceae
[10,11]. In Pseudomonas aeruginosa, the most common mechanisms
of carbapenem resistance are oprD porin mutations, rather than
production of carbapenemases [9]. When carbapenemases are pro-
duced, MBLs are the most commonly associated with P. aeruginosa.
Why Is It Important to Detect Carbapenemase-Producers?
Detection and/or characterization of carbapenemase production
is no longer required by the Clinical and Laboratory Standards
Institute (CLSI) as of 2010. It was at this time that the carbape-
nem breakpoints were lowered for the Enterobacteriaceae, result-
ing in the removal of the recommendation for routine testing for
the detection of carbapenemase production among isolates with
elevated carbapenem MICs. With implementation of the 2010
carbapenem breakpoints, the interpretations for the carbapenem
antibiotics are reported as tested regardless of the mechanism
of carbapenem resistance [12]. With the introduction of novel
β-lactam combination agents, such as ceftazidime-avibactam or
meropenem-vaborbactam, that have activity against specific classes
of carbapenemases and not others (neither has activity against
MBLs, for example), it has become increasingly important from
the perspective of patient management to differentiate CP from
non-CP CRO and, more specifically, to identify the class of car-
bapenemase being produced. This allows antimicrobial steward-
ship teams to stratify patients who may be prioritized for use of
the newer agents. Identification of CP from non-CP CRO can
also determine whether an agent may be considered empirically
pending the completion of antimicrobial susceptibility testing (i.e.,
12 Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier
if an MBL producer is detected, it rules out the use of the newer
β-lactam combination agents).
The Centers for Diseases Control and Prevention (CDC) rec-
ommends screening isolates for carbapenemase production when
carbapenem-resistant Enterobacteriaceae (CRE) are encountered to
facilitate prevention and control of carbapenem resistance [13]. If
a carbapenemase producer is detected, additional infection pre-
vention measures are taken to prevent its spread in the hospital
setting, such as placing the patient on contact precautions and/or
staff and patient cohorting. Therefore, laboratories may choose to
continue to detect carbapenemase producers, as it remains impor-
tant for infection control and antimicrobial stewardship, as well
as epidemiological purposes, to monitor rates of CP CRO and to
identify when new variants of carbapenemases are introduced into
the health care system.
Thus, it is becoming increasingly important to delineate the
mechanism of carbapenem resistance among CRO for clinical
care. Due to these clinical needs, we have observed the introduc-
tion of both phenotypic and genotypic methods for the detection
of carbapenemase production among Gram-negative organisms.
This article describes the various phenotypic methods available
to detect carbapenemase production by cultured organisms and
the considerations when choosing a method to implement based
on the resources of a specific institution.
In Brief: Genotypic Methods for Detection of
Carbapenemases
Due to the nature and scope of this article, we do not cover in
depth genotypic methods for detection of carbapenemase produc-
tion, which include both laboratory-developed and automated/
commercial nucleic acid amplification tests (NAT), microarrays,
and next-generation sequencing (NGS) applications [5,14]. It
should be briefly noted, however, that molecular methods, includ-
ing nucleic acid amplification, generally necessitate additional
equipment, such as a thermocycler, and may require the purchase
of additional instrumentation for microarray and NGS applica-
tions. While PCR is a routine method in most clinical laborato-
ries, the major limitations of molecular-based methods include
the inability to detect novel genes and variants, as they require a
priori knowledge of the specific targets for detection. Home-brew
NAT, microarrays, and NGS also require significant molecular
expertise, whereas automated sample-to-answer NAT platforms
require minimal expertise. The Cepheid (Sunnyvale, CA, USA)
Xpert Carba-R assay is the only automated FDA-cleared molecu-
lar in vitro diagnostic test that is able to detect the most common
carbapenemase genes (blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP)
from cultured isolates, with a reported sensitivity of 100% and
specificity of 97% [15]. It is also FDA cleared for detection of
gastrointestinal colonization from rectal swabs [15]. The Gene-
POC (Québec, Canada) Carba Test and the BD (Wagengingen,
Netherlands) MAX Check-Points Check CPO (CE marked) are
two other automated molecular platforms that detect the five most
common carbapenemase genes, blaKPC, blaNDM, blaVIM, blaOXA-48,
and blaIMP; these are not yet FDA cleared. Despite the relatively
short turnaround time (TAT) of 1 to 3 hours for these automated Growth-Based Phenotypic Assays
systems, the main disadvantage is the cost per test. Phenotypic tests that are growth based evaluate the presence of
carbapenemases by detecting the growth of an organism in the
Phenotypic Tests: What Are Your Options? presence of an antibiotic and provide next-day results. Examples
of such tests are the MHT and the CIM or mCIM. The MHT,
Several types of phenotypic tests for the detection of carbapen-
the original phenotypic method described to detect carbapene-
emase production continue to be developed and include growth-
mase production among CRE, involves plating of a carbapenem-
based methods (modified Hodge tests [MHT] [16] and the
susceptible Escherichia coli strain with a carbapenem disk (usually
carbapenem inactivation method [CIM]/modified CIM [mCIM]
meropenem) in the center and plating linear streaks of the car-
[17]), colorimetric assays (Carba NP and derivatives) [18], matrix-
bapenem-resistant test isolate away from the carbapenem disk [21].
assisted laser desorption ionization–time of flight mass spectrom-
The test is interpreted as positive if there is enhanced growth of
etry (MALDI-TOF MS)-based tests [19], and lateral-flow assays
the carbapenem-susceptible indicator strain toward the carbape-
(LFA) [5,20]. For the most part, these assays, in their original
nem disk along the linear streak of the test isolate, resulting in a
form, detect carbapenemase production broadly and do not further
cloverleaf-like indentation (Fig. 2A) [5,21]. The MHT, while well
identify or differentiate carbapenemase classes (class A, B, or D)
known to most laboratories and relatively simple to perform, is no
or specific enzymes (KPC, NDM, OXA-48, etc.). However, most
longer endorsed by the CLSI as of 2018, in favor of newer methods
methods have been further adapted with class-specific inhibitors
with better performance characteristics, including the mCIM and
to identify the various Ambler classes of carbapenemases.
the Carba NP [12,22]. The MHT is prone to false-positive results
when ESBL or AmpC producers are concurrently present with
The performance characteristics of the various phenotypic assays
porin mutations and/or efflux, as well as false-negative results with
vary by Ambler class of carbapenemase and by the Gram-negative
SME, OXA-type, and most notably MBL carbapenemases (such
organism being evaluated (Table 1) [2]. In addition to the perfor-
as NDM-1) [2]. This lack of sensitivity and specificity coupled
mance characteristics of the test, other factors, such as cost, ease of
with the subjective nature of interpreting the results has allowed
use, TAT, technical expertise required, and equipment, can make
newer methods to supplant the MHT in the guidelines. Anecdot-
the implementation of a test more or less advantageous depending
ally, however, it continues to be used in several laboratories during
on the needs of the implementing laboratory. Several factors need
this transition period due to the above-mentioned ease of use and
to be considered on a case-by-case basis, including the prevalence
familiarity with the method.
of CRO in the population served by the laboratory and the cost
and utility of the test once implemented (Fig. 1). Various options The mCIM, the newer of the two growth-based methods discussed
in phenotypic tests are available, allowing laboratories to choose here, requires broth inoculation of the carbapenem-resistant test
a method that best suits their specific needs, as described below. isolate in the presence of a meropenem disk for 4 hours in tryptic

Table 1. Sensitivities and specificities of the Carba NP, mCIM, MALDI-TOF MS hydrolysis, and lateral-flow methods by organism and
carbapenemase classa

Carba NP mCIM MALDI-TOF MS hydrolysis Lateral flow methods

[2,9,29,49-51,66] [2,9,23-26] [19,39,52-57] [20,47,48,58,59]

Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity

Enterobacteriaceae 100 99-100 94-100 95-100

Class A 72-100 98-100 100 100
Class B 89-100 98-100 80-100 100
Class D 40-100 100 92-100 100

Pseudomonas aeruginosa 98-100 80-100 100 100

Class A 67-100 100
Class B 77-100 89-99 97-100
Class D

Acinetobacter baumannii 100 53-100 100 100

Class A
Class B 60-83 57-90 100
Class D 8-39 18-79 100
a
Only studies conducted with a sample size of at least 30 were included for statistical validity.

Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier 13

soy broth, followed by placement of the disk onto Mueller-Hin-
ton agar (MHA) streaked with a carbapenem-susceptible E. coli
indicator strain, with overnight incubation. The test works on
the premise that carbapenemase producers will rapidly hydrolyze
the meropenem in the disk during the 4-h incubation period so
that when the disk is removed and plated with the carbapenem-
susceptible indicator strain, the disk has reduced or no activity and
will yield a zone diameter between 6 and 15 mm after overnight
incubation. Non-CP CRE will not hydrolyze the meropenem disk
efficiently during the 4-h incubation period and will remain active
against the indicator strain, yielding zone diameters of ≥19 mm
after overnight incubation (Fig. 2B) [17].
The mCIM has been further developed for detection of car-
bapenemase production among carbapenem-resistant P. aerugi-
nosa by increasing the inoculum required for reliable detection
of carbapenemase production to a 10-µl loopful compared to the
1-µl loopful for Enterobacteriaceae [9]. It is not recommended for
detection of CP in Acinetobacter baumannii due to poor perfor-
mance characteristics (Table 1). A newer variant of the test using
Tris-HCl buffer for extraction (CIMTris) detects carbapenemase
production in Acinetobacter and Pseudomonas spp. with a sensitiv-
ity of 98% and a specificity of 93% [23]. To further differentiate
MBL from serine carbapenemases, the mCIM has been further
adapted by adding ethylenediaminetetraacetic acid (EDTA), a cat-
ion chelator and inhibitor of MBLs. The EDTA mCIM (eCIM)
is performed simultaneously with the mCIM, and a ≥5-mm zone
diameter difference for the E. coli indicator strain between the
eCIM and the mCIM is indicative of MBL production, while a
≤4-mm difference indicates production of a serine carbapenemase
that is not inhibited by the addition of EDTA (Fig. 2B). Differ-
entiation of MBL from serine carbapenemases is clinically useful,
as newer β-lactam combinations have activity against serine car-
bapenemases but not MBL producers, as mentioned previsously.
Overall, the mCIM displays excellent sensitivity and specificity of
≥98% [2,9,23-26]. Based on our experience, the specificity of the
assay may be slightly lower than originally described, as Entero-
bacter cloacae isolates lacking known carbapenemase enzymes iden-
tified by whole-genome sequencing may produce false-positive
mCIM results. However, it has shown impressive sensitivity, even
among isolates harboring carbapenemases that may test suscep-
tible to the carbapenems (unpublished data). It is in general a very
cost-effective method, which only requires standard laboratory
supplies found in most microbiology laboratories and is simple to
set up and interpret. The biggest limitation of the mCIM test is
the TAT of 18 to 24 hours (Table 2). Thus, if same-day results are
required, perhaps due to the lack of isolation rooms, among other
reasons, the mCIM may not be the ideal method for an institution.
The mCIM is a modified version of the original CIM initially
described in 2015 by van der Zwaluw et al. [17] and is the best-
known modification of the CIM. The mCIM utilizes a 1-µl loop-
ful of the CRE instead of a 10-µl loopful for setup and substitutes

Choosing a Phenotypic Carbapenemase Detection Method

Testing Considerations

1. Turn-around time (TAT) 6. Regulatory status of test

2. Costs (+, ++, +++) 7. Required equipment
3. Testing volumes 8. Required reagent preparation
4. Organisms to be tested 9. Carbapenemases detected
5. Ease-of-use 10. Performance characteristics

Yes Are same day results required? No

Testing modalities to consider: Testing modalities to consider:

1. MALDI-TOF MS carbapenem hydrolysis methods (+*) 1. MHT (+)
2. Combined commercial AST/phenotypic CP detection panels (+++) 2. mCIM derivatives (+)
3. Lateral Flow Tests (+++) 3. Combination disk methods (++)
4. Colorimetric methods (manual [+*] and commercial [++ to +++]) derivatives 4. Gradient diffusion methods (+++)
• Combination disk
methods
No Ability to detect all carbapenemases? Yes Readily available laboratory supplies? No
• Gradient diffusion
methods

Lateral Flow Antigen Tests Ease-of-use Yes

• Rapid (15 min)
• Simple
• Simplex or multiplex test
for detection of KPC,
NDM, VIM, IMP and/or
OXA-48-like
Increased difficulty/expertise required Simple
• MHT
• mCIM
• MALDI-TOF MS hydrolysis methods • Commercial colorimetric tests
require alterations to instrument settings • Combined AST/phenotypic
• Manual Carba NP methods requiring AST panels
frequent reagent preparation

Figure 1. Algorithm for selecting a phenotypic method for detection of carbapenemase-producing carbapenem-resistant organisms.
+, <$1.00; ++, ≤$1.00 to $5.00; +++, >$5.00; *, can be affected by volume.

14 Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier

tryptic soy broth in place of water for better detection of MBL
producers that require divalent cations for activity, and the
length of the incubation was extended to 4 hours from 2 hours
for enzymes with reduced hydrolytic activity (such as OXA-type
enzymes) during the meropenem disk inactivation step compared
to the CIM [27]. Additional modifications of the CIM have been
recently published, including a CIMplus test that allows detection
of carbapenemase in 8 hours [28] and the rapid carbapenem inacti-
vation method (rCIM), which brings the carbapenemase detection
time down to less than 3 hours [29]. The CIMplus test involves
simultaneous incubation of the meropenem disk with a 10-µl loop-
ful of the tested strain and streaking of an MHA plate with a sus-
ceptible E. coli indicator strain, which permits early growth of the
E. coli indicator strain to allow a more rapid interpretation of the
CIM results when the disks are placed on the plate after 2 hours of
incubation with the tested strain [28]. They also describe simulta-
neous setup of the CIMplus test with EDTA (a class B inhibitor)
and another with phenyl boronic acid (PBA; a class A inhibitor) to
further differentiate between class A, B, and D enzymes (through
a process of elimination). The rCIM method involves using two
10-µl loopfuls of bacteria suspended in sterile water with two 10-µg
meropenem disks for 30 min at 37°C, after which the suspension
is centrifuged and the supernatant is added to a broth culture of
the E. coli indicator strain and monitored for growth using a neph-
elometer every 30 min for 2 hours [29]. These modifications allow
the possibility of same-day interpretation of results, overcoming
the biggest disadvantage of the CIM test. Similar modifications
are likely to be described for the mCIM version.

Figure 2. Phenotypic methods for the detection of carbapenemase-producing carbapenem-resistant organisms. (A) Modified Hodge test.
(B) mCIM and eCIM results. (C) Carba NP and variants. Manual CLSI Carba NP positive result. Tube a, no imipenem added, red, Tube b. imipenem
added, yellow. RAPIDEC© CARBA NP (bioMérieux, Inc) positive result. Well d. No imipenem added, red. Well e. Imipenem added, yellow. Manual
Blue Carba positive result. Tube a, imipenem added, yellow, Tube b, no imipenem added, blue Rapid CARB Blue Screen kit (Rosco Diagnostica)
positive result. Tube a, imipenem added, yellow. Tube b, no imipenem added, blue.

Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier 15

 Table 2. Characteristics of select phenotypic tests for the detection of carbapenemase production among carbapenem-resistant Gram-negative organisms

16
Valuea

Manual
Commercial Carba NP Carba NP CLSI and Modified Hodge test CIM/mCIM and BD Phoenix CPO LFA MALDI-TOF MS hydrolysis Molecular methods
Characteristic methods [2,67] variants [2] [2] ­ ariants [2]
v Detect [46] [20, 47,48] [40] [40]
Anticipated False negatives False negatives False positives Rare false False positives False positives Adding NH4HCO3 Only detects targeted
false negatives with OXA-48 type, with OXA-48 type, with ESBL/AmpC positives with E. with AmpC; false with OXA-163 and or ZnSO4 increases genes; IMP targets
and false mucoid isolates, or mucoid isolates, or and permeability cloacae harboring negatives with OXA-405, which detection of class D are usually specific to
positives isolates with weak isolates with weak defects; false multiple non- KPC, IMP, and VIM are considered and class B enzymes, IMP-1.
hydrolytic activity hydrolytic activity negatives with SME, carbapenemase low-activity respectively.
MBL, OXA types mechanisms carbapenemases
but considered
false positives

Cost per test ++ to +++ + + + +++ +++ + +++

(USD)b
Carbapenem- Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae, All Gram-negative
resistant and Pseudomonas and P. aeruginosa and P. aeruginosa (to date) (to date) P. aeruginosa and A. organisms
organisms aeruginosa baumannii
CarbAcineto, CIMTris,

Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier

Acinetobacter spp. P. aeruginosa and
A. baumannii
Equipment Most or all pH meter Standard laboratory Standard BD Phoenix All reagents in kit MALDI-TOF MS At a minimum, a
reagents in kits supplies laboratory instrument instrument thermocycler; other
supplies instrumentation is
required for microarray
and NGS-based
methods.
Limitations Color change Color change Reading of results Required initial Requires the Reagents must be Requires the Inability to detect
subjective subjective can be subjective. setup and then instrumentation used immediately instrumentation be in novel genes and
plating of disk be in place if opened; place variants
Frequent reagent
onto lawn of E. coli
preparation due unopened reagents Detection range (m/z) of
to short shelf life stable up to 2 yr. 160-600 (different from
of imipenem- Inability to detect bacterial ID); incubation
containing solution times or lysis steps vary.
novel enzymes
Interpretation Color change from Color change from Enhanced growth Zone of inhibition Included in Presence or absence Spectra of intact Amplification curves;
of results red to orange/ red to yellow of the carbapenem- BD Phoenix of a line on the carbapenem and its colorimetric detection
yellow (Rapidec susceptible strain susceptibility LFA where the degradation products of hybridization events
CARBA NP; Neo- toward the panel results antibodies capture for microarrays, whole
Rapid Carb screen); carbapenem disk the epitope genome sequencing
color change from along the linear of the specific assembly and analysis
blue to green to streak of the test carbapenemase
yellow (Rapid Carb isolate, resulting
Blue Screen) in a cloverleaf-like
indentation

TAT 30 min-2 h 30 min-2 h 18 h-24 h 18 h-24 h <36 h 5 min-15 min 30 min-4 h 1-3 h for automated
NAT to >48 h for NGS

Regulatory RUO LDT; CLSI and LDT; no longer CLSI LDT; mCIM, CLSI RUO RUO LDT RUO; except the
status RAPIDEC CARBA: EUCAST endorsed endorsed endorsed; CIM, Cepheid CARBA-R is
FDA cleared EUCAST endorsed FDA cleared for isolates
and rectal swabs
a
Modified from Tamma et al., 2017 [2].
b
USD, U.S. dollars; +, <$1.00; ++, ≤$1.00 to 5.00; +++, >$5.00; Cost can be affected by volume.
Colorimetric Carbapenemase Assays
Colorimetric assays have been developed to detect carbapenemase
production directly from bacterial culture isolates, providing same-
day results [5]. They can detect all Ambler classes of carbapen-
emases by using a pH indicator, which results in a color change
(phenol red, red to yellow; bromothymol blue, blue to yellow)
when imipenem is hydrolyzed by carbapenemases, producing an
acidic product (Fig. 2C) [5]. The Carba NP is the first described
assay of this kind and was initially endorsed by both the CLSI and
the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) for use with the Enterobacteriaceae, P. aeruginosa, and
A. baumannii. Studies have since shown that the CLSI Carba NP
method has poor detection of carbapenemase production in A.
baumannii, which prompted the removal of the Carba NP from
CLSI guidelines in 2018 as an officially sanctioned method for A.
baumannii [9, 30]. The main advantage of the Carba NP test is the
capability to provide same-day results, though frequent reagent
preparation can encumber its implementation due to waste and
associated costs if the reagents are not used within the specified
shelf life (72 hours for the imipenem-containing solution), espe-
cially if the volumes of the laboratory may not support this prac-
tice [31]. The test has demonstrated good sensitivity and excellent
specificity for detection of carbapenemase producers (Table 1).
However, false-negative results can occur with OXA-48-like
enzymes or when mucoid carbapenemase producers are tested [2].
Furthermore, some enzymes with weak hydrolytic activity may
result in minor color changes, making interpretation subjective,
or may yield false-negative results.
Since the initial description of the Carba NP in 2012 by P. Nord-
mann and colleagues, several modifications have been described.
These modifications include changes to the extraction reagents,
the inoculum, the starting pH, the pH indicators, and the read-
ing times, as well as methods with Ambler class-specific inhibi-
tors to further differentiate carbapenemase classes (Carba NP II)
[5,22,32,49]. Due to low-level outer-membrane permeability and
slow hydrolysis of imipenem by class D OXA-type carbapenemases
common to Acinetobacter, further changes to manual Carba NP
tests were required for detection of CP Acinetobacter spp. One
method, the CarbAcineto NP, which targets carbapenemase pro-
duction in Acinetobacter spp., was described and reported to have a
sensitivity of 89 to 95% after increasing inoculum sizes and using
NaCl in place of a lysis buffer [32,33].
Several commercial assays have also been developed, including
the RAPIDEC Carba NP (bioMérieux, Marcy L’Etoile, France)
[34], which is FDA cleared for use with the Enterobacteriaceae and
P. aeruginosa, and the research use only (RUO) Neo-Rapid Carb
Screen [35], Rapid Carb Blue Screen (RoscoDiagnostica A/S,
Taastrup, Denmark), and β CARBA NP (Bio-Rad Laboratories
N.V., Marnes-la-Coquette, France). The commercial assays were
designed with the aim of simplifying testing by providing premade
reagents with longer shelf life to remove the need for reagent prep-
aration [36]. The RAPIDEC Carba NP performs particularly well,
with a reported sensitivity and specificity of 97% [37]. Although
some of the modifications have resulted in increased sensitivity
and ease of use, they share, for the most part, the same limitations
described above for the original Carba NP, with false-negative
results occurring with OXA-48-like enzymes or mucoid isolates
and the subjective nature of interpreting minor color changes for
enzymes with weak hydrolytic activity.
MALDI-TOF MS Methods
MALDI-TOF MS-based hydrolysis tests detect the presence of
carbapenemases by assessing for the presence of hydrolysis of a
carbapenem (ertapenem, imipenem, or meropenem) using the
detection of mass peaks associated with the hydrolyzed carbape-
nem products when a CP CRO is incubated with a carbapenem
antibiotic for a specific length of time [38]. The sensitivity of
MALDI-TOF-based methods in detecting OXA-48-like and
MBL producers can be improved by adding NH4HCO3 or
ZnSO4, respectively [19,39]. The major limitation of MALDI-
TOF hydrolysis-based methods is the necessity of having a mass
spectrometer already in place and a mass detection range that is
different from that required for bacterial identification, as well as
the lack of standardization of methods (incubation times and/or
lysis steps) (Fig. 1) [40]. MALDI-TOF MS hydrolysis methods
have also been further adapted, with class-specific inhibitors using
PBA to detect class A enzymes and dipicolinic acid to detect class
B enzymes by inhibition of the hydrolysis reaction in the presence
of the inhibitors [41]. The TAT for this method can range from
30 minutes to 4 hours.
Other methods have been developed to detect carbapenemases
using MS technologies and peak identification methods. MALDI-
TOF MS can show a specific and distinct peak of a protein that
is associated with a carbapenemase-bearing plasmid, such as the
blaKPC-bearing pKpQIL plasmid [42]. To date, this method has
been described only for this specific plasmid-associated peak.
Additionally, a liquid chromatography–tandem-MS method that
can detect unique tryptic peptides of the KPC protein in clinical
isolates has been shown to be both a rapid and promising test, with
sensitivity and specificity of 100% [43].
Carbapenemase Class-Distinguishing Tests
In addition to broad phenotypic carbapenemase detection meth-
ods, several class-specific tests have been described. These tests
include some discussed above, such as the eCIM, Carba NP II, and
MALDI-TOF MS hydrolysis inhibitor-based methods. Gradient
diffusion strips have been developed including a combination of
a β-lactam and β-lactam/β-lactamase inhibitor on opposite ends
of a single strip. The MICs of the β-lactam individually and in
combination with the β-lactamase inhibitor are read, and a pre-
set ratio of the two or the presence of a phantom zone indicates
a positive result. These tests have been developed for detection
of KPC producers using boronic acid and of MBLs using EDTA
or 2-mercaptopropionic acid. The MBL and KPC gradient strips
have reported sensitivities and specificities of >92% and >94%,
respectively [44]. A major limitation of these tests is the cost (~$5/
strip) of a narrowly based test that is specific for detection of a par-
ticular class of carbapenemase. The MASTDISCS combi Carba
plus disc system D73C (MAST-Carba plus; Mast Group, Bootle,
Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier 17
Merseyside, United Kingdom) is another recently developed test
for identification of carbapenemase types produced by Enterobac-
teriaceae isolates. It contains five disks: faropenem alone (disk A)
and combined with MBL inhibitors (disk B), KPC inhibitors (disk
C), and AmpC inhibitors (disk D) and temocillin combined with
MBL inhibitors (disk E). It is able to distinguish between MBL,
KPC, and OXA-48 producers, as well as AmpC producers with
porin loss, with a specificity of 91% [45]. Interpretation of the
results can be complex, especially when multiple carbapenemases
are produced.
Lastly, the BD Phoenix CPO Detect (BD Diagnostics Systems,
Sparks, MD) is the first automated carbapenemase test that is
combined with a routine Phoenix antimicrobial susceptibility test-
ing (AST) panel with computer-assisted algorithm-based detec-
tion [46]. The test allows simultaneous AST determination and
carbapenemase detection and classification (similar to ESBL tests
on automated systems). It has shown high sensitivity of >97% for
carbapenemase detection and a specificity of 69% and is able to
distinguish between the different carbapenemase classes, with sen-
sitivities of 85% for class A, 72% for class B, and 89% for class
D enzymes [46].
Lateral-Flow Assays
Immunochromatographic methods, such as LFA, have also been
recently developed for the phenotypic detection of carbapen-
emases. These assays use nanoparticles bound to a nitrocellulose
membrane with antibodies to capture epitopes specific to car-
bapenemase enzymes within the lateral-flow device [20,47]. Tests
such as the OXA-48-K-Se T, KPC-K-SeT assays (Coris BioCon-
cept), and NDM LFA have been commercially developed for easy
and rapid detection of OXA-48, KPC, and NDM carbapenemases.
These assays have shown excellent performance characteristics
in a few studies to identify OXA-48, KPC, and NDM producers
directly from cultured bacterial isolates and positive blood cultures
[20,47]. The most exciting development in this arena is a multiplex
lateral-flow immunoassay, the Carba5, for the rapid identification
of NDM, KPC, IMP, VIM-type, and OXA-48-like carbapen-
emases in Enterobacteriaceae [48]. The Carba5 LFA can detect the
big 5 carbapenemases, similar to the automated molecular assays
mentioned above. However, it is likely to be cheaper; costs have
not yet been established, but similar tests have been found to be
cost-effective [48]. It also does not require any specialized equip-
ment or trained staff [48]. These assays are not yet FDA cleared.
Performance Characteristics
Performance of the available carbapenemase detection methods
differ by the Ambler class of carbapenemase and also between the
Enterobacteriaceae, P. aeruginosa, and A. baumanii. Generally speak-
ing, all the classes of carbapenemases, A, B, and D can be found in
all three groups of organisms. For the purposes of this review, we
included only the sensitivities and specificities of studies conducted
with a sample size of at least 30 for statistical validity. The Carba
NP test has variable sensitivity but performs best in the Enterobac-
teriaceae, particularly among Class B carbapenemases (MBLs), with
sensitivity ranging from 89 to 100% and specificity approaching
18 Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier
100% [2,29,49-51]. In contrast, Carba NP performs poorly in A.
baumannii, with sensitivities of 60 to 83% and 8 to 39% for class
B and class D carbapenemases, respectively [9] (Table 1). In P.
aeruginosa, the Carba NP performs moderately, with a sensitivity
range of 67 to 100% in class A carbapenemases and 77 to 100% in
class B carbapenemases [9] (Table 1). The specificity with Carba
NP for both A. baumannii and P. aeruginosa is 100%.
With regard to sensitivity and specificity, the mCIM method per-
forms comparably to or better than the Carba NP. The mCIM has
a sensitivity of ≥98% for all classes of carbapenemases in Enterobac-
teriaceae and sensitivities of 100% in class A carbapenemases and
98 to 99% in class B carbapenemases in P. aeruginosa [2,9,23-25].
Similar to the Carba NP, the mCIM also has poor performance
characteristics with A. baumannii (Table 1) [23].
MALDI-TOF MS hydrolysis methods perform far better in Aci-
netobacter spp. than the other methods evaluated here, including
the Carba NP and mCIM, with a sensitivity and specificity of
100% in both class B and D carbapenemases [52,53] (Table 1).
MALDI-TOF hydrolysis detects class B carbapenemases for P.
aeruginosa with a sensitivity of 97 to 100% and a specificity of
100%, and has also been evaluated for all the classes in Enterobac-
teriaceae, with sensitivities of 100% in class A, 80 to 100% in class
B, and 92 to 100% in class D carbapenemases and overall speci-
ficity of 94 to 100% [19,53-57] (Table 1).
Performance characteristics of the LFA for the detection of
carbapenemases have also been described among the Entero-
bacteriaceae, with a sensitivity of 100% for class A, B, and D car-
bapenemases and specificities of 95 to 100% [20, 48, 58, 59]
(Table 1). The global performance of the Carba5 multiplex assay
specifically reached a sensitivity of 100% in the five target-related
enzymes among the Enterobacteriaceae, with a specificity of 95%
[48]. Despite the lack of studies done to evaluate LFA in detect-
ing CP P. aeruginosa and Acinetobacter spp., tests with non-CP
isolates of these bacteria have been done to demonstrate specific-
ity of 100%.
What Method Should You Choose?
General considerations
Choosing an appropriate phenotypic test for carbapenemase detec-
tion in a laboratory is dependent on a number of factors. TAT,
cost, testing volumes, organisms being tested, ease of use, regula-
tory status of the test, equipment, and reagents, as well as perfor-
mance characteristics, are all important considerations (Fig. 1).
The prevalence of carbapenemases in a population can help deter-
mine how frequently to perform testing and whether active sur-
veillance is warranted. Different patient care areas (i.e., inpatient
wards, outpatient clinics, and long-term care settings) may war-
rant different algorithms or surveillance practices, depending on
prevalence and risk. Several options are available, allowing labo-
ratories to choose a method that best suits their needs. The pros
and cons of these tests are summarized in Table 2.
Infection control
The infection control procedures of the hospitals, clinics, or long-
term/post-acute care settings the laboratory serves should play a
role in determining the type of testing needed. The CDC recom-
mends health care facilities consider ongoing evaluation to deter-
mine the incidence of CRE organisms among patients seen in their
facilities and, if carbapenemase testing is not available through
their laboratories, identify outside laboratories with the capacity
to perform testing when necessary [13]. In facilities that use con-
tact precautions, systems should be developed and implemented to
identify patients with a history of CRE colonization or infection
in order to place them on contact precautions, and laboratories
should have a protocol to notify clinical or infection control teams
when CRE and/or CP CRE are identified from either clinical or
surveillance cultures [13]. In settings where identification of CP
CRE is a prerequisite for instituting contact precautions, shorter
TAT may be an important factor. In our facility, if an organism
does not meet our MDR definition (resistant to all but 2 classes of
antimicrobials) and is a non-CP CRE, then contact precautions are
deemed unnecessary. TAT may be especially important in facilities
that may not have access to private rooms for all comers, as earlier
results can allow triage of private isolation rooms.
If same-day results are required for infection control purposes,
testing modalities to consider include MALDI-TOF MS hydro-
lysis methods, combined commercial AST/phenotypic CP detec-
tion panels, lateral-flow tests, and colorimetric methods (manual
or commercial) (Fig. 1). Of these, the lateral-flow antigen tests
are more limited in their ability to detect all carbapenemases and
may be more useful in settings where a targeted approach to car-
bapenemase detection may be more practical if a limited spectrum
is prevalent in the population. On the other hand, in the context
of wider public health surveillance or outbreak investigation, the
ability to detect all types of carbapenemases is crucial. Generally
speaking, MALDI-TOF MS hydrolysis and manual Carba NP
methods require greater technical expertise to perform, with the
MALDI-TOF MS requiring alterations to instrument settings
and the manual Carba NP methods requiring frequent reagent
preparation. In contrast, the commercial colorimetric tests and
the combined AST/phenotypic panels are simpler to perform.
Antimicrobial stewardship
Effective January 2017, the Joint Commission announced a new
antimicrobial stewardship standard for hospitals, critical-access
hospitals, and nursing care centers [60]. Laboratories should work
closely with the antimicrobial stewardship representatives for their
facilities to determine the appropriate test to consider for antimi-
crobial stewardship initiatives. It is important for the laboratory
to work in conjunction with the antimicrobial stewardship team
in deciding which tests to implement, as well as in assisting clini-
cians with appropriate interpretation of tests for carbapenemase
production. Several novel antimicrobial drugs, both in use and in
the pipeline, have activity against some classes of carbapenemases
but not others. For example, the novel carbapenem combination
agent meropenem-vaborbactam, approved in 2017 by the FDA
for use in complicated urinary tract infections caused by CREs,
has potent inhibitory activity against KPC-producing serine
β-lactamases; however, it does not have activity against class D
carbapenemases (OXA-48 like) or MBLs (NDM, VIM, and IMP)
[61-63]. Another novel combination agent, ceftazidime-avibactam,
was also FDA approved as of 2014, and evidence suggests it may
have a role to play in treating MDR CRE and P. aeruginosa infec-
tions. The β-lactamase inhibitor avibactam has inhibitory activity
against serine carbapenemases, including class A (KPC) and some
class D (OXA-48) enzymes, but like vaborbactam, it does not have
activity against MBLs, as it lacks a serine residue in the active site
[64,65]. As more niche antimicrobials enter the pipeline toward
approval, rapid methods for detection of carbapenemase produc-
tion and the carbapenemase class will play increasingly important
roles in guiding antimicrobial choice and clinical management
decision making.
Microbiology laboratory considerations
In addition to the considerations listed above, the volumes of
tests, instrumentation available, and expertise are among the other
considerations for the microbiology laboratory. If the volume of
CRO encountered at a facility is not high (less than 5 tests a week),
then commercially available tests (combined AST/phenotypic CP
detection panels, commercial colorimetric tests, or LFA) or tests
requiring readily available supplies (MHT or mCIM) are ideal for
these scenarios. If same-day results are not required, the MHT,
mCIM, combination disk methods, and gradient diffusion meth-
ods are all testing modalities that can be considered. The MHT
and mCIM are both relatively cheap to perform compared to the
commercial alternatives and use readily available laboratory sup-
plies. For laboratories with more extensive resources or higher vol-
umes of testing, the manual Carba NP variants or MALDI-TOF
MS hydrolysis methods become additional options to consider.
Method Verification for Clinical Use
Once a test has been chosen based on the particular needs of the
institution and the microbiology laboratory, verification must be
performed prior to clinical testing. Methods that are FDA cleared
or are endorsed by CLSI have undergone previous validation test-
ing (defining the performance characteristics of the assay). Thus,
verification (confirmation of established performance character-
istics by the personnel who will perform the testing) of the FDA-
cleared or the CLSI-endorsed laboratory-developed test (LDT)
prior to implementation for clinical care is required. A verifica-
tion plan for these tests will require that precision (reproducibil-
ity) and accuracy be evaluated. At a minimum, 20 CP organisms
(expected positive) and 10 non-CP CRO (expected negative)
should be tested, with at least 90% agreement with the expected
results. Isolates from the CDC and FDA Antibiotic Resistance
Isolate Bank (https://wwwn.cdc.gov/arisolatebank/Panel/AllIso-
late) can be requested and used for the verification. These isolates
have been previously molecularly characterized, and the molecular
results can be used as the comparator (expected) results for the
verification. The CPO chosen for the verification should include
various Gram-negative organisms and carbapenemase variants
covering the big five carbapenemase classes, KPC, NDM, VIM,
IMP, and OXA-48-like enzymes (see the CDC and FDA Antibiotic
Clinical Microbiology Newsletter 41:2,2019 | ©2019 Elsevier 19
Resistance Isolate Bank Enterobacteriaceae Carbapenemase Diversity
Panel and Pseudomonas aeruginosa Panel). For precision, inter-assay
and intra-assay reproducibility should be determined by testing a
minimum of three CP-CRO and three non-CP-CRO (negative
results) over 3 days (inter-assay) and on the same day (intra-assay)
with expected ≥95% qualitative precision. Assays that are not FDA
cleared or that are modifications to existing tests may require fur-
ther validation to establish the performance characteristics prior
to implementation in the clinical laboratory.
Summary
At the moment, detection of carbapenemase production among
Gram-negative organisms is not required by CLSI for the manage-
ment of patients provided the 2010 carbapenem breakpoints are
implemented. However, detection of carbapenemase production
is becoming increasingly important for epidemiological, infec-
tion prevention, and control and antimicrobial stewardship ini-
tiatives, requiring laboratories to consider further delineation of
the mechanism of carbapenem resistance among CRO for clinical
care. Due to these clinical needs, we have observed the introduc-
tion of both phenotypic and genotypic methods for the detection
of CP CRO. When considering a test for detection of CP CRO,
phenotypic methods are generally more affordable than molecular-
based assays. There are several options for phenotypic tests that
differ in TAT, costs, ease of use, equipment required, carbapen-
emases detected, and performance characteristics. The phenotypic
method best suited for a particular laboratory or hospital system is
based on the needs of and resources available to the institution and
microbiology laboratory. Once a method is chosen, verification or
validation of the assay should be undertaken prior to implementa-
tion for clinical use.
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