2.

6 Carbapenemase Detection Methods

Over the period, several methods were developed for detection of

carbapenemase producing organisms. These methods can be classified into

phenotypic and genotypic methods.

2.6.1 Phenotypic methods

Antibiotic Susceptibility test (AST) is considered as conventional phenotypic method

for detection of carbapenemases producing organisms. This is normally done using

automated systems like Microscan, Vitek2 and phoenix. Manual systems such as

Minimum inhibitory concentration (MIC), Modified hodge test, E test, broth micro

dilution agar, Carba NP, Carbapenem in activation method and chromogenic based

media.

2.6.1.1 Manual systems

Carba NP test is a colorimetric assay developed by Nordmann et al. (2012)

(NORDMANN; POIREL; DORTET, 2012) and proven to be faster with a lower

false-positive rate than MHT. It detects carbapenemase producing organisms

from the change of Ph of phenol red to orange due to hydrolysis of imipenem by

carbapenemase(PASTERAN; TIJET; MELANO; CORSO, 2015).

Carbapenem inactivation method (CIM) which measures the diameter of the

zone of inhibition of E. coli ATCC 25922 to determine the carbapenemase activity

of the tested bacteria after the carbapenem disk is inactivated by the test

bacterium. The modified CIM became very useful in microbiology laboratories. It

was recommended by the CLSI in 2017 due to its cost effectiveness, simplicity

and availability with a 100% sensitivity and specificity. (KUCHIBIRO; KOMATSU;

YAMASAKI; NAKAMURA et al., 2018)

The matrix-assisted laser desorption ionization–time of flight mass spectrometry

(MALDI–TOF MS) method was introduced by Hrabak et al. in 2011(HRABÁ K;

WALKOVÁ ; ŠTUDENTOVÁ ; CHUDÁ ČKOVÁ et al., 2011) and has been very useful

for the screening of carbapenemase producing organisms. It operates by

detecting and measuring the by-products of the hydrolysed carbapenem when

the extracted proteins of bacteria is incubated with meropenem or ertapenem.

(HRABÁ K; WALKOVÁ ; ŠTUDENTOVÁ ; CHUDÁ ČKOVÁ et al., 2011) It can also be

used to identify many pathogens based on their chemical composition.

Minimum inhibitory concentration (MIC) makes used of different platforms

including broth micro dilution agar, E-test and Microscan, vitek and phoenix. It

is regarded as the most reliable indicator to detect Carbapenem resistance.

(EUCAST 2019)(AL-ZAHRANI, 2018).

Double disk synergy test is used for confirmatory detection of extended

spectrum beta-lactamases using third generation cephalosporins and

Clavulanate. The later, clavulanate will do as beta-lactamase inhibitor and if the

3rd generation cephalosporins produces ESBL, it will produce zone of inhibition

toward clavulanate and give a unique shape on agar plate e.g., Muller Hinton

Agar

Moreover, in line with the recommendation of CLSI, Isolated Enterobacteriaceae

demonstrating resistance to third generation cephalosporins, should undergo a

confirmatory step such as the Modified Hodge Test (MHT) or the Xpert® Carba-R

platform (KPC, NDM, VIM, IMP, OXA-48) by Cepheid

(https://www.cepheid.com/en/tests/Healthcare-Associated-Infections/Xpert-Carba-R )(AL-

ZAHRANI, 2018)
A Positive Modified Hodge Test is shown by clover leaf-like cavity of Escherichia

coli susceptible strains growing along the tested organism growth streak within the

disk diffusion zone (PANDURANGAN; BEGUMESAK; NARAYANASAMY,

2015) meanwhile a negative Modified Hodge Test shows no growth of E. coli along

the tested organism growth streak within the disc diffusion zone.(PANDURANGAN;

BEGUMESAK; NARAYANASAMY, 2015)

2.6.1.2 Automatic systems

VITEK 2 is an integrated modular automated system which provides rapid and

accurate identification and susceptibility testing results for most clinical isolates

including carbapenemase producing enterobacteriaceae. Identification is made on the

basis of biochemical reactions and MIC determinations are made by applying an

algorithm to the growth kinetics monitored by the VITEK 2 system.

The BD Phoenix is an automated system designed and marketed by Becton Dickinson

Diagnostic Systems (Sparks). Introduced in 2003 for bacterial identification and AST.

The Phoenix AP instrument automatically adjusts the turbidity of the CPE suspension

to a 0.5 McFarland standard, prepares a dilution for susceptibility testing and adds

indicator to the susceptibility testing inoculum. This instrument uses racks that can

hold identification (ID) and antimicrobial susceptibility testing (AST) broths required

to set up isolates. Isolate are monitored every 20 min by colorimetric and fluorescence

readings for identification and antimicrobial susceptibility testing is based on redox

and turbidimetric detection of growth. (Richter and Ferraro 2007; Jorgensen and

Ferraro 2009).

MicroScan WalkAway is another automated system which can process 96 panels at

the same time. It uses LabPro data management system on a contiguous computer.
MicroScan WalkAway uses a pH indicator and fluorogenic substrates to detect CPE

enzymatic activity. (Ellner and Myers 1981; Rodriguez et al. 1999).

Although phenotypic tests, vary in their sensitivity, they are widely used due to

their simplicity and low cost. [6] Alternatively, screening method to detect CRE,

could be done with chromogenic based media.[6]

2.6.2 Genotypic methods

The molecular techniques for CRE detection is used as a reference method to confirm

the result of the phenotypic methods by screening for the presence of carbapenem

hydrolysing genes

Multiplex-PCR, used for differentiating the MBL genes of five different families

(SPM, IMP, VIM, GIM, SIM) in a single PCR run such as (KPC,NDM,VIM,IMP and

OXA-48).[12]

Verigene ® is a non-amplified molecular approach used for gram negative blood

culture by extracting the DNA from the positive blood culture, follow by Microarray

analysis. (https://www.luminexcorp.com/the-verigene-system/)[14]

Microfluidic chip technology which allows the rapid detection of pathogens and their

resistance genes (Kim et al., 2017) was used to detect carbapenem-resistance genes,

with high sensitivity and specificity (both >90.0%), and fully met the requirements for

clinical diagnoses (Zhang G. et al., 2018).

FilmArray® BCID is a multiplex PCR that identifies 24 microorganisms commonly

encountered in blood, including Gram-positive bacteria, Gram-negative bacteria, and

Candida spp. additionally, the test targets one carbapenemase gene, blaKPC, along

with the Gram-positive resistance markers, mecA, vanA and vanB.

Based on the algorithm for the detection of carbapenemase (Figure 2), the most

comprehensive method used to detect carbapenemase genes is probably the Whole

Genom Sequencing (WGS) tool which provides a detailed description of all genes

located in the organism [14]