Table 2.

The advantages and limitations of the current and emerging detection techniques for the most common carbapenemases (carbapenem-hydrolyzing enzymes).

Techniques	Advantages	Limitations
Culture-based methods	Simple and cost-effective	Time-consuming (>24 h)
1. Improved AST tests: E-test or disk diffusion test [32,107,108]	Detect KPC and MBLs with good sensitivity (>82%) and specificity (>95%)	Insufficient for OXA-48 Require specific reagents and pure culture
2. Modified Hodge Test (MHT) * [108,110]	Detects KPC with good sensitivity (>69%) and specificity (>90%)	Insufficient for MBLs Requires pure culture
3. Carbapenem-inactivation methods (CIM) * [107,108]	Detect all carbapenemases with higher sensitivity (>90%) and specificity (>95%)	Require pure culture
4. Selective media: SUPERCARBA, Colorex KPC, ID Carba, CHROM agar KPC, etc. [112,113,114]	Detect carbapenemases from direct patient samples SUPERCARBA has higher sensitivity (>96.5%)	Variable sensitivity (40–96.5%) and specificity (>50%)
Rapid phenotypic methods	Rapid (<24 h)	Costly equipment
1. Colorimetric assay: CarbaNP test and its automated kits * [60,107,108]	Detect carbapenemases with good sensitivity (>70%) and specificity (>80%) Simple, rapid (<2 h), and cost-effective No equipment requirement	Insufficient for OXA-48 Require pure culture
2. MALDI-TOF MS * [123,125,126]	Rapidly (1–4 h) detects KPC and MBLs with good sensitivity (>72.5%) and specificity (>95%) Low-measurement cost and simple	Requires data analysis Insufficient for OXA-48 Requires single isolated colonies
3. Emerging techniques: BCDA, FC, microfluidic techniques, and Raman spectroscopic techniques [116,119,120,122,123]	Simple and rapid (<4 h) Good sensitivity (>80%) and specificity (>90%) from pure culture	Lower applicability on specimens Insufficient work on carbapenemases
Genotypic methods	Rapid and highly specific (>90%) and sensitive (>90%)	Costly and complex equipment
1. PCR-based methods: qPCR, RT-PCR, mPCR, automated PCR (Xpert system, Check-Direct, and Carba-R-assay) [123,131,135] *	Gold standard and rapid (<4 h) Detect and type all carbapenemases directly from specimens	High technical requirements and specific reagents High measurement cost
2. Loop-mediated isothermal amplification (LAMP) [123,142]	Simple and moderate cost Applicable in low-resource settings	Specific reagents and complex primer design
3. Whole genome sequencing (WGS) [123,141] *	Discovers a new resistance mechanism	Longer turn-around time Complex data management
4. Emerging techniques: FISH, microarray techniques, PCR-ESI-MS, and NucliSENS EasyQKPC [116,123,143]	Rapid (<6 h) Detect carbapenemases	Require specific equipment and reagents Insufficient work on carbapenemases
Immunological Methods Enzyme-linked immunosorbent assay (ELISA), an Immunochromatographic assay [99,123,138,151]	Rapid and moderate cost Poor sensitivity and specificity directly from specimens	Complex and difficult antibody design due to antigenic site modification
Biosensors: Emerging Technology	Rapid, Simple, and Cost-effective	Specific Equipment
1. Electrochemical assays: Impedimetric, potentiometric, and voltammetric [43,156,160] 2. Optical assays: Raman scattering, SPR, and SERS [118,120,138,161]	Detect carbapenemases Moderate cost	Require equipment for signal processing and data analysis Insufficient work on AMR and carbapenemase detection from pure culture and specimens
2.1. Plasmonic biosensors [167,172]	Rapid, simple, and cost-effective Detect carbapenemases with good sensitivity (78%) and specificity (97%) No equipment requirement	Insufficient work on AMR and carbapenemase detection from pure culture and specimens

* Techniques have been used in diagnostic laboratories (clinical and public health laboratories). AST: antibiotic susceptibility test, MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, BCDA: bioluminescence-based detection assays, FC: flow cytometry, FISH: fluorescence in situ hybridization, PCR-ESI-MS: PCR amplification coupled with electrospray ionization mass spectrometry, NucliSENS EasyQKPC: RNA-targeted molecular approach, SPR: surface plasmon resonance; SERS: Surface-Enhanced Raman Scattering technique.