Recent Advances and Future Perspectives in the Detection of Carbapenem-Resistant Acinetobacter Baumannii

Abstract

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major global public health threat. The World Health Organization (WHO) has classified CRAB as a critical priority pathogen. This threat is mainly attributed to its multidrug resistance, efficient nosocomial transmission, and limited therapeutic options. Timely and accurate detection is essential for early diagnosis, targeted antimicrobial therapy, and effective infection control. This review summarizes recent advances in CRAB detection technologies, including conventional phenotypic methods, nucleic acid amplification-based assays, genomic and AI-assisted diagnostics, mass spectrometry-based methods, and biosensor platforms. Conventional antimicrobial susceptibility testing (AST) remains the reference standard. However, it requires bacterial culture and isolation before testing, and the process usually takes 18–24 h after bacterial colony growth. This delay may limit its value for early therapeutic decision-making. Phenotypic carbapenemase assays provide complementary information on carbapenemase activity and resistance mechanisms, but their sensitivity, specificity, and clinical applicability vary across methods. Molecular methods substantially shorten turnaround times, with PCR and qPCR generally producing results within approximately 2–3 h. Isothermal amplification platforms further accelerate detection. LAMP-based assays report turnaround times of approximately 21–60 min, whereas RPA-based assays complete detection within approximately 40–90 min, supporting their potential application in point-of-care testing (POCT). Emerging genomic approaches, including whole-genome sequencing, nanopore sequencing, and metagenomic sequencing, enable comprehensive resistance profiling, outbreak investigation, and surveillance, while AI-assisted diagnostics may enhance resistance prediction and clinical interpretation. Mass spectrometry-based methods enable high-throughput species identification and can support resistance profiling in selected assay formats, with some assays detecting resistance-associated features within 15–90 min. Biosensor-based and CRISPR-integrated platforms offer highly sensitive, rapid, and portable detection, although challenges remain in multiplexing, standardization, cost, and clinical validation. Overall, this review highlights the transition of CRAB diagnostics from culture-based susceptibility testing toward integrated, automated, and clinically adaptable platforms, emphasizing the need to balance analytical performance, turnaround time, cost, and suitability for POCT.

Introduction

Acinetobacter baumannii (A. baumannii) is a Gram-negative, aerobic, non-fermentative bacillus with remarkable environmental adaptability. Its ability to survive on dry surfaces for prolonged periods and form biofilms has made it one of the most important opportunistic pathogens responsible for healthcare-associated infections. A. baumannii primarily affects immunocompromised patients, especially those in intensive care units (ICUs). It can cause pneumonia, bloodstream infections, wound infections, and other severe infections associated with high morbidity and mortality.^1–3 The increasing prevalence of carbapenem-resistant Acinetobacter baumannii (CRAB) has become a major global public health concern.⁴ The clinical management of CRAB remains challenging due to limited therapeutic options, efficient nosocomial transmission, and high mortality, particularly in ICU settings. Notably, a recent multicenter study involving 393 critically ill patients with CRAB bloodstream infections reported a 28-day mortality rate of 56.5%.⁵

Carbapenems have long been regarded as a cornerstone of treatment for multidrug-resistant Gram-negative bacterial infections. Although the advent of novel agents such as sulbactam-durlobactam and cefiderocol has expanded treatment options for CRAB infections,⁶ CRAB remains a major global public health threat. Consequently, the World Health Organization (WHO) has designated CRAB as a critical-priority pathogen requiring urgent research and intervention.^7,8 Genomic surveillance has shown that CRAB is dominated by several international clonal lineages, with IC2 being the most widely disseminated worldwide, although the distribution of clones and carbapenemase determinants varies across geographical regions.⁴ The widespread dissemination of CRAB is driven by multiple resistance mechanisms. These include the production of OXA-type carbapenemases and metallo-β-lactamases, overexpression of RND-type efflux pumps, altered outer membrane permeability, and horizontal transfer of antimicrobial resistance determinants mediated by mobile genetic elements.^8,9

Timely identification of CRAB is essential for guiding targeted antimicrobial therapy, implementing infection control measures, and preventing hospital outbreaks. Among conventional phenotypic AST techniques, broth microdilution serves as the gold standard. Nevertheless, these routine assays require a lengthy turnaround of 24–48 h, potentially hindering timely targeted treatment at the onset of infection.^10,11 In response, numerous rapid diagnostic technologies have been developed, including PCR-based assays, isothermal amplification techniques, mass spectrometry-based methods, biosensor platforms, and, more recently, genomic sequencing and artificial intelligence (AI)-assisted diagnostic approaches. The substantial differences in analytical performance, turnaround time, cost, and equipment requirements highlight the need for systematic comparison of these technologies in clinical and laboratory settings.

Although previous studies have summarized different CRAB detection methods, many reports remain largely descriptive, and integrated comparisons of diagnostic performance, clinical applicability, POCT suitability, and translational limitations are still needed. Moreover, rapid advances in emerging technologies, such as droplet digital PCR, polymerase spiral reaction, whole-genome sequencing, nanopore sequencing, metagenomic sequencing, CRISPR-based detection, and AI-assisted diagnostics, have significantly expanded the landscape of CRAB detection and warrant an updated and integrated assessment.

In this review, we summarize recent advances in CRAB detection technologies, including phenotypic methods, molecular assays, mass spectrometry-based approaches, biosensor platforms, and emerging genomic and AI-assisted diagnostics. Particular emphasis is placed on comparing diagnostic performance, clinical applicability, suitability for POCT, and current limitations. Future trends toward integrated, automated, and precision diagnostic systems are also discussed. By providing a critical overview of existing strategies and emerging technologies, this review aims to inform the development of next-generation CRAB diagnostic platforms and support clinical decision-making and infection control in the context of antimicrobial resistance. Ultimately, the clinical value of diagnostic technologies depends not only on analytical performance but also on their impact on patient outcomes, including timely initiation of effective therapy and infection control.

Current Status of Antimicrobial Resistance in A. baumannii and its Resistance Mechanisms

A. baumannii is widely distributed in both environmental and healthcare settings and is frequently detected on surfaces that come into direct contact with patients. A study conducted in a hospital in Xuhui District, Shanghai, reported the highest contamination rate among patient-contact items (34.56%), followed by shared-use items (26.84%).¹² In 2021, CRAB was among the antimicrobial-resistant pathogens associated with the highest attributable mortality worldwide, ranking second only to methicillin-resistant Staphylococcus aureus (MRSA).¹³ According to the 2024 annual report of the China Antimicrobial Surveillance Network (CHINET), A. baumannii remained one of the five most frequently isolated bacterial species. Among these isolates, 22,255 CRAB isolates showed high levels of antimicrobial resistance. Resistance rates to the carbapenems imipenem and meropenem were 99.6% and 99.7%, respectively (Figure 1).

Figure 1.

Antimicrobial resistance of 22255 carbapenem-resistant Acinetobacter baumannii strains (%).¹⁴

Analysis of resistance trends from 2005 to 2024 showed that CRAB resistance rates to imipenem and meropenem declined in 2024 compared with previous years but remained high (Figure 2). This sustained high-level resistance underscores the complexity of CRAB resistance mechanisms.

Figure 2.

Variation of resistance of Acinetobacter baumannii to imipenem and meropenem (2005–2024).¹⁴

The resistance mechanisms of CRAB are complex and diverse, including carbapenemase production, efflux pump overexpression, target site modification, and reduced outer membrane permeability.⁸ As illustrated in Figure 3, these mechanisms collectively contribute to high-level carbapenem resistance and pose significant challenges for clinical treatment. Recent studies have shown that CRAB resistance is also associated with modulation of quorum sensing systems and activation of efflux pumps, which together enhance bacterial survival under stress conditions and facilitate the dissemination of resistance genes.⁷ Notably, among these mechanisms, carbapenemase production is considered one of the most clinically significant mechanisms contributing to carbapenem resistance. OXA-type carbapenemases, particularly blaOXA-23, blaOXA-24/40-like, and blaOXA-58, are widely distributed among clinical CRAB isolates. Metallo-β-lactamases, encoded by genes such as blaNDM, blaIMP, and blaVIM, have also emerged as important contributors to carbapenem resistance.⁸ In addition, overexpression of RND-type efflux pump systems, including AdeABC, AdeFGH, and AdeIJK, reduces intracellular antibiotic accumulation and enhances resistance. Alterations or loss of outer membrane proteins further contribute to this process.⁹ The dissemination of these resistance determinants is frequently mediated by mobile genetic elements, including insertion sequences, transposons, integrons, and plasmids, facilitating horizontal gene transfer and the spread of carbapenem resistance genes.¹⁵

Figure 3.

Mechanisms of carbapenem resistance acquisition in Acinetobacter baumannii.

Conventional Phenotypic Methods

Conventional Antimicrobial Susceptibility Testing Methods

Conventional antimicrobial susceptibility testing (AST) remains the cornerstone for phenotypic detection of CRAB. Among currently available methods, broth microdilution (BMD) is widely recognized as the reference standard for antimicrobial susceptibility testing. It provides quantitative minimum inhibitory concentration (MIC) values with high accuracy and reproducibility. In this method, bacterial isolates are exposed to serial dilutions of antimicrobial agents, and susceptibility is interpreted according to established Clinical and Laboratory Standards Institute (CLSI) breakpoints. Despite its reliability, BMD is labor-intensive and requires standardized laboratory procedures, limiting its practicality for routine high-throughput testing.^10,11

The Kirby-Bauer disk diffusion (K-B) method is one of the most widely used AST approaches in clinical laboratories. Compared with BMD, it is simpler, less expensive, and easier to perform. However, because susceptibility is inferred from inhibition zone diameters rather than direct MIC determination, its ability to discriminate isolates with borderline susceptibility may be limited.¹⁶ The gradient diffusion method (Etest) combines the principles of diffusion and dilution methods and enables direct MIC determination using a predefined antimicrobial gradient. This method generally demonstrates good agreement with reference susceptibility testing methods and provides quantitative results that are useful for clinical decision-making. However, its relatively high cost may limit routine implementation in resource-constrained settings.¹⁶ To improve laboratory efficiency, the VITEK 2 automated antimicrobial susceptibility testing system has been introduced for routine clinical use.¹⁷ It provides rapid and standardized susceptibility testing with minimal hands-on time and is well suited for high-throughput clinical laboratories. Previous studies have shown generally good agreement among the K-B method, Etest, and VITEK 2 for susceptibility categorization in A. baumannii. Nevertheless, VITEK 2 yielded lower MIC₅₀ and MIC₉₀ values than the Etest in the evaluated isolates.^16,17

Phenotypic Carbapenemase Detection Methods

Phenotypic carbapenemase assays are based on the hydrolysis of carbapenem antibiotics by carbapenemases. Commonly used methods include the Modified Hodge test (MHT), the Carba NP test, and the modified carbapenem inactivation method (mCIM).^18,19 The MHT detects carbapenemase production by observing a characteristic cloverleaf-shaped growth pattern of an indicator strain around a carbapenem disk. Although the assay is simple and inexpensive, it is highly subjective and prone to false-positive results. Owing to its poor specificity and limited reliability, its clinical use has markedly declined with the availability of improved alternative assays.¹⁸ The Carba NP test is a rapid colorimetric assay that detects carbapenem hydrolysis through pH-dependent color changes of a phenol red indicator. Compared with other phenotypic carbapenemase assays, the Carba NP test offers excellent specificity and can generate results within a few hours. However, its sensitivity for A. baumannii is limited. In a multicenter evaluation, the specificity reached 100%, whereas sensitivity was only 18.8%, indicating a substantial risk of false-negative results.¹⁹ The mCIM determines carbapenemase production by assessing the inactivation of a carbapenem disk following incubation with the test isolate. Compared with the Carba NP test, mCIM demonstrated higher sensitivity but lower specificity for the detection of carbapenemase-producing A. baumannii. In the same evaluation, the reported sensitivity and specificity of mCIM were 79.8% and 52.9%, respectively. Given its relatively low specificity, mCIM may produce false-positive results and should preferably be interpreted together with molecular or complementary phenotypic methods rather than used alone for definitive confirmation of carbapenemase production.¹⁹

Phenotype-based methods vary considerably in their diagnostic performance and practical applicability. Conventional AST methods provide susceptibility profiles but do not directly assess carbapenemase activity, whereas carbapenemase assays directly evaluate enzyme activity and may therefore offer additional information regarding resistance mechanisms. Among conventional AST methods, BMD remains the reference standard because of its high accuracy, while automated systems such as VITEK 2 improve laboratory efficiency and throughput. In contrast, carbapenemase detection assays exhibit distinct performance characteristics; the Carba NP test provides rapid results with excellent specificity but limited sensitivity, whereas mCIM generally demonstrates higher sensitivity at the expense of specificity. Consequently, no single phenotypic method is optimal for all clinical scenarios. Method selection should therefore be guided by diagnostic objectives, laboratory capacity, and turnaround time requirements. A comparative summary of representative phenotypic detection methods is presented in Table 1.

Table 1.

Comprehensive Comparison of Phenotype-Based Detection Methods for CRAB

Method	Sensitivity	Specificity	Turnaround Time (h)	Cost	Equipment Requirement	Clinical Applicability	POCT Suitability
BMD10,11	Reference standard	Reference standard	18-24	Low	Incubator, microtiter plates	Reference method; accurate but labor-intensive	No
K-B16	High agreement	High agreement	18-24	Very low	Incubator, MH agar plates, antibiotic disks	Routine screening; simple and low cost	No
Etest16	High agreement	High agreement	18-24	High	Incubator, MH agar plates, E-test strips	MIC determination; useful for confirmation	No
VITEK17	Moderate-high agreement	Moderate-high agreement	6-18	High	Automated VITEK system	Automated, high-throughput routine testing	No
MHT18	Variable	Low	18-24	Low	Incubator, MH agar plates, meropenem disks, indicator strain	Historical carbapenemase assay; limited current use	No
Carba NP19	Low	Excellent	0.5-2	Low-moderate	Incubator, colorimetric reagents	Rapid carbapenemase assay; low sensitivity in A. baumannii	Limited
mCIM19	Moderate	Low	18-24	Low	Incubator, MH agar plates, meropenem disks	Carbapenemase screening; simple but false positives possible	No

Notes: For antimicrobial susceptibility testing (AST) methods, sensitivity and specificity reflect agreement with reference susceptibility testing methods rather than true diagnostic sensitivity and specificity. Turnaround time refers to the assay time required after isolation of bacterial colonies.

Nucleic Acid Amplification Technologies

PCR-Based Molecular Detection Techniques

Polymerase chain reaction (PCR) is a classical nucleic acid amplification technique that enables rapid detection through the amplification of specific genetic targets using sequence-specific primers. Conventional PCR and real-time quantitative PCR (qPCR) have been used for the detection of carbapenemase-encoding genes in CRAB, including blaOXA-23, blaOXA-40, and blaOXA-51.^20–22 Compared with phenotypic methods, these molecular approaches directly identify antimicrobial resistance genes and provide more rapid confirmation of carbapenem resistance mechanisms. In a comparative study, both conventional PCR and qPCR achieved complete detection of carbapenemase-producing A. baumannii isolates, with approximate turnaround times of 3 h and 2 h, respectively.²⁰ Nevertheless, PCR-based detection depends on prior knowledge of target genes and may fail to identify resistance mechanisms that are not included in the assay design.

Multiplex PCR allows the simultaneous amplification of multiple resistance-associated targets in a single reaction. By combining several primer sets within one assay, this approach reduces the need for separate single-target reactions and facilitates the rapid screening of clinically relevant resistance determinants.²³ However, assay performance depends on primer specificity and requires careful optimization when multiple targets are included. Multiplex real-time qPCR further integrates simultaneous target detection with real-time fluorescence monitoring, enabling rapid molecular characterization of resistance determinants while improving detection efficiency.²⁴

Droplet digital PCR (ddPCR) represents a further advancement in PCR technology by partitioning reaction mixtures into thousands of picoliter-sized droplets, allowing absolute quantification of target nucleic acids without the need for standard curves.^25,26 In CRAB detection, a duplex ddPCR assay targeting blaOXA-23 and gltA demonstrated a lower limit of detection than the corresponding qPCR assay. This finding highlights its potential for detecting low-abundance targets in clinical specimens. Despite its promising analytical performance, the routine application of ddPCR remains limited by the requirement for dedicated digital PCR platforms and more complex workflows.²⁶

In addition to resistance detection, PCR-based approaches such as M13-PCR, repetitive extragenic palindromic PCR (REP-PCR), and arbitrarily primed PCR (AP-PCR) have been applied to the molecular typing of CRAB isolates.^27,28 These methods are mainly used for strain discrimination, epidemiological surveillance, and outbreak investigation rather than routine detection of carbapenem resistance. REP-PCR has been reported to show good concordance with pulsed-field gel electrophoresis (PFGE) while requiring substantially less time and laboratory effort.²⁸

Isothermal Amplification Techniques

Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) is one of the most widely studied isothermal amplification methods for the rapid detection of A. baumannii and CRAB. Because LAMP runs under isothermal conditions, it does not require thermal cycling and can be performed with relatively simple equipment. This makes it useful for rapid screening and resource-limited settings. Multiplex LAMP assays can detect species-specific and resistance-associated targets in the same reaction, enabling simultaneous pathogen identification and antimicrobial resistance screening. One multiplex LAMP assay generated results within approximately 21 min, supporting its value for timely clinical decision-making.²⁹ Different readout formats have further improved the practical applicability of LAMP. When combined with nucleic acid fluorescence lateral flow assays (NFLFA), LAMP enables visual result interpretation without sophisticated instruments, thereby enhancing its suitability for POCT. A LAMP-NFLFA platform completed detection within 43 min while maintaining operational simplicity.³⁰ RealAmp, by contrast, combines isothermal amplification with real-time fluorescence monitoring in a closed-tube format. This design reduces contamination risk and simplifies result interpretation. Although portable RealAmp devices can provide results in less than 60 min, they still require dedicated fluorescence detection equipment.³¹

Several challenges limit the wider application of LAMP. The need for multiple primers makes assay design complex and may increase non-specific amplification if the reaction is not carefully optimized. In addition, the high amplification efficiency of LAMP can increase the risk of carryover contamination and false-positive results. Therefore, LAMP is well suited for rapid screening, but reliable clinical use still depends on careful assay design and strict quality control.

Recombinase Polymerase Amplification

Recombinase polymerase amplification (RPA) is another promising isothermal amplification approach for CRAB detection. Compared with LAMP, RPA works at lower temperatures and requires less instrumentation, making it attractive for portable and decentralized testing. Among RPA-based platforms, RPA-LFS assays are particularly suitable for POCT, outbreak investigation, and resource-limited settings because they can generate results within approximately 40 min while requiring minimal laboratory infrastructure.³² However, the simplicity of this approach may limit its suitability for comprehensive resistance profiling. Therefore, RPA-LFS is generally better suited for targeted detection of a limited number of markers rather than broad resistance characterization.

Multiplex detection remains a major challenge for RPA-based diagnostics. Interactions among multiple primer-probe sets may reduce amplification efficiency and compromise specificity. To address this issue, CRISPR-assisted detection systems have been combined with RPA. RPA-CRISPR-Cas12a integrates rapid amplification with crRNA-guided target recognition. This combination improves analytical specificity and reduces the risk of false-positive results. Although this platform has a more complex workflow than conventional RPA-LFS, it can complete detection within 90 min and offers greater potential for multiplex analysis.³³

From a practical perspective, LAMP-based platforms currently appear to be more mature for routine CRAB screening because they have established workflows and diverse readout formats. In contrast, RPA-based assays offer better portability and lower equipment requirements, making them attractive for decentralized testing. The integration of CRISPR technology with RPA may further expand the role of isothermal amplification in future POCT applications by improving specificity and multiplexing capability. The key characteristics of these nucleic acid amplification-based methods are summarized in Table 2.

Table 2.

Comparison of Nucleic Acid Amplification-Based Methods for CRAB Detection

Method	Detection Limit	Turnaround Time (min)	Cost	Equipment Requirement	Clinical Applicability	POCT Suitability
PCR20,21	3×10−1 ng/μL	180	Moderate	Thermocycler, imaging system	Confirmatory testing	No
qPCR20,22	1×10−3 ng/μL	120-180	High	Real-time PCR instrument	Routine clinical diagnosis	No
Multiplex qPCR24	10 CFU/assay	NR	High	Real-time PCR instrument	Simultaneous gene detection	No
ddPCR26	1×10−4 ng/μL	180-300	High	Digital PCR instrument	Low-abundance target detection	No
Multiplex LAMP29	1×102 CFU/μL	21	Low	Incubator, fluorescence reader	Rapid screening	Limited
LAMP-NFLFA30	199 CFU/mL	43	Low	Incubator, lateral flow strip	Resource-limited settings	Yes
RealAmp31	1×103 CFU/mL	<60	Moderate	Incubator, real-time fluorescence reader	Resource-limited settings	Limited
RPA-LFS32	1-10 CFU/reaction	40	Low	Incubator, Lateral flow strip	Resource-limited settings	Yes
RPA-CRISPR-Cas12a33	1.3×10−6 ng/μL	<90	Moderate	Incubator; UV lamp	Resource-limited settings	Yes

Notes: Cost was categorized as low, moderate, or high according to instrument and assay complexity.

Abbreviations: NR, not reported; POCT, point-of-care testing.

Polymerase Spiral Reaction

Polymerase spiral reaction (PSR) is a potentially relevant emerging isothermal nucleic acid amplification technique that employs a strand-displacing DNA polymerase and a single pair of specially designed primers to achieve rapid amplification under isothermal conditions. Compared with LAMP, PSR requires fewer primers and involves a simpler primer design process. The reaction is typically performed at 61–65 °C and can be completed within 30–60 min. Amplification products may be detected by fluorescence or colorimetric methods.³⁴ Although PSR has not yet been applied specifically to CRAB detection, its technical features suggest potential value as a future alternative for rapid molecular diagnostics in resource-limited settings.

Emerging Genomic and Intelligent Diagnostic Technologies

Whole-Genome Sequencing

Whole-genome sequencing (WGS) is a high-resolution genomic approach that enables comprehensive characterization of bacterial genomes through determination of the complete DNA sequence of an organism. Unlike targeted molecular assays that detect only predefined resistance genes, WGS provides simultaneous insights into antimicrobial resistance determinants, virulence factors, mobile genetic elements, phylogenetic relationships, and bacterial population structure. Consequently, WGS has emerged as a powerful tool for the characterization, surveillance, and epidemiological investigation of CRAB.³⁵

The utility of WGS in elucidating the molecular epidemiology and resistance mechanisms of A. baumannii has been demonstrated in recent studies. Large-scale genomic analyses have enabled comprehensive characterization of the resistome, virulome, and mobilome of clinical isolates.³⁶ Furthermore, WGS has revealed the critical role of insertion sequences, transposons, and other mobile genetic elements in facilitating the acquisition and dissemination of antimicrobial resistance genes.³⁷ These findings have substantially improved our understanding of the evolution and transmission dynamics of CRAB in healthcare settings. In addition to resistance profiling, WGS enables high-resolution phylogenetic analysis for outbreak investigation and transmission tracking, making it valuable for monitoring the dissemination of high-risk CRAB clones.³⁸ Despite its advantages, the routine clinical implementation of WGS remains constrained by relatively high costs, prolonged turnaround times, and the need for specialized bioinformatics expertise.

Nanopore Sequencing

Nanopore sequencing is a third-generation sequencing technology that enables real-time nucleic acid analysis through the detection of ionic current changes generated as DNA or RNA molecules pass through nanopores. Compared with conventional short-read sequencing platforms, this technology produces substantially longer reads. These long reads enable characterization of antimicrobial resistance genes (ARGs), plasmids, insertion sequences, and other mobile genetic elements associated with carbapenem resistance. Its portability and rapid data generation have promoted its application in antimicrobial resistance surveillance and clinical diagnostic workflows.³⁹ Recent studies have shown that nanopore sequencing can provide timely pathogen identification and resistance profiling directly from clinical specimens.⁴⁰ The ability to resolve complex genomic regions also facilitates reconstruction of resistance islands and mobile genetic elements, which is valuable for investigating the evolution and transmission of CRAB.⁴¹ Although sequencing accuracy and bioinformatics requirements remain important limitations, ongoing technical improvements continue to expand the potential of nanopore sequencing in CRAB detection and epidemiological surveillance.

Metagenomic Sequencing

Metagenomic next-generation sequencing (mNGS) is an untargeted sequencing approach that enables comprehensive analysis of all nucleic acids present in clinical specimens without prior culture or target-specific amplification. Unlike conventional molecular methods, mNGS can simultaneously identify pathogens, ARGs, and microbial community composition, making it particularly useful for diagnosing mixed infections and culture-negative cases.⁴² mNGS-based resistome profiling enables characterization of resistance determinants associated with CRAB and prediction of resistance to multiple antimicrobial classes, highlighting its value for resistome profiling and antimicrobial stewardship.⁴³ In addition, metagenomic sequencing can identify resistance-associated mobile genetic elements, providing insights into the dissemination of resistance genes and the epidemiology of carbapenem resistance.⁴⁴

Compared with conventional culture methods, mNGS offers broader pathogen coverage and higher pathogen detection rates, particularly in critically ill patients.⁴⁵ However, its clinical application remains constrained by sequencing costs, bioinformatics complexity, and the imperfect correlation between genotypic resistance determinants and phenotypic susceptibility. Ongoing advances in sequencing technologies and bioinformatics pipelines are expected to further enhance the role of mNGS in CRAB diagnosis and epidemiological surveillance.

AI-Assisted Diagnostics

Artificial intelligence (AI) has emerged as a promising tool for improving the detection and diagnosis of antimicrobial-resistant pathogens, including CRAB. By analyzing complex genomic, microbiological, and clinical datasets, AI algorithms can support resistance prediction and clinical decision-making.⁴⁶ Machine learning algorithms can identify antimicrobial resistance determinants from genomic and phenotypic datasets, enabling rapid antimicrobial resistance prediction and supporting personalized antimicrobial therapy.⁴⁷ Beyond resistance prediction, AI has the potential to improve diagnostic workflows by integrating sequencing data with clinical information, thereby facilitating earlier and more informed therapeutic interventions for CRAB infections.⁴⁸ However, its clinical implementation remains limited by challenges related to data quality, model interpretability, and the need for large, well-annotated datasets. As sequencing technologies and computational methods continue to advance, the integration of AI with genomic sequencing and point-of-care diagnostic platforms is expected to enhance the speed, accuracy, and clinical applicability of CRAB diagnostic approaches while supporting antimicrobial stewardship.⁴⁹

Mass Spectrometry Techniques

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a microbial identification technique based on microbial protein profiling.⁵⁰ Microbial samples are co-crystallized with a chemical matrix and ionized by laser irradiation. The resulting ions are separated according to their mass-to-charge ratio in a time-of-flight analyzer. The obtained spectral profiles are then matched against reference databases for rapid microbial identification.

The high discriminatory capability of MALDI-TOF MS enables reliable identification at the species level and, in some cases, discrimination among closely related species. It is therefore well-suited for the accurate identification of CRAB and other members of the Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex, including A. pittii and A. nosocomialis.^51,52 MALDI-TOF MS also allows rapid detection of colistin resistance in A. baumannii within 15 min by detecting phosphoethanolamine modification of lipid A.⁵³ MALDI-TOF MS-based detection of carbapenem hydrolysis in CRAB relies on enzyme activity rather than gene detection. This approach enables rapid assessment of carbapenemase activity and may provide additional insights into resistance mechanisms. This approach is suitable for large-scale screening and has the potential to become a standardized assay in microbiology laboratories for detecting A. baumannii and other carbapenemase-producing bacteria.

Liquid Chromatography-Tandem Mass Spectrometry

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) combines the high separation efficiency of liquid chromatography with the high sensitivity and specificity of tandem mass spectrometry. Samples are separated on a chromatographic column according to differences in their partitioning between the stationary and mobile phases. After chromatographic separation, precursor ions are selected and fragmented, and the resulting product ions are analyzed for qualitative and quantitative characterization.^54,55 Compared with PCR-based assays, LC-MS/MS provides a complementary approach for resistance profiling by directly detecting resistance-associated proteins. This capability may improve the assessment of antimicrobial resistance phenotypes. LC-MS/MS has been used to simultaneously detect multiple β-lactamases, and their expression levels show high concordance with corresponding resistance phenotypes.⁵⁵ In contrast, MALDI-TOF MS usually requires separate assays for different enzymes, which increases assay time and cost. LC-MS/MS requires complex sample preparation, including protein precipitation, extraction, and concentration, to minimize matrix effects and interference from impurities. By contrast, MALDI-TOF MS generally involves simpler sample preparation, usually requiring only that the sample be mixed with a matrix and spotted onto a target plate. For carbapenemase activity assays, samples may be incubated with a carbapenem substrate before MALDI-TOF MS analysis.

Mass spectrometry can rapidly identify A. baumannii and characterize its drug resistance with high throughput. However, novel biosensor platforms provide complementary merits: simple sample pretreatment, portable devices, and great potential for POCT.

Biosensor Technologies

Biosensors convert signals generated during biological recognition processes into measurable electrical or optical outputs through physical or chemical transducers. These platforms facilitate rapid A. baumannii detection and provide new approaches for early diagnosis and infection control.⁵⁶ Nucleic acid aptamer- and nanomaterial-based biosensors enable rapid, sensitive, and highly specific detection of A. baumannii. Their analytical performance can be further enhanced by integrating surface plasmon resonance, electrochemical impedance sensing, and fluorescence-based detection.^57–59 A plasmonic biosensor based on glucan-functionalized nanoparticles and oligonucleotide probes targeting the blaKPC gene has been developed, enabling quantitative detection by absorbance spectroscopy and differentiation between target and non-target samples within 30 min.⁵⁸ More notably, CRISPR-based biosensors improve detection sensitivity through efficient signal amplification. Their key advantage lies in the collateral cleavage activity of CRISPR effector nucleases, such as Cas12a and Cas13a, which enables signal amplification after target recognition. This amplification strategy provides innovative tools for the clinical diagnosis of antimicrobial resistance in A. baumannii.⁶⁰ Recent technological advances have led to the development of RPA-CRISPR assays with fluorescence and lateral-flow readouts, electrochemical sensing platforms, and microfluidic integration. These methods are capable of detecting carbapenemase genes including blaOXA-23. They exhibit high sequence specificity and single-base discrimination, with robust diagnostic accuracy validated in clinical samples. Future antimicrobial resistance gene detection is expected to focus on amplification-free direct detection,⁶¹ fully automated microfluidic platforms,⁶² and live-cell imaging-based dynamic monitoring.⁶³ Despite challenges related to multiplex detection capability and standardization,⁶⁴ advances in enzyme engineering and AI-guided crRNA design are expected to make CRISPR-Cas systems a promising platform for precision diagnosis of CRAB. To meet practical application requirements, biosensors should be further optimized to improve their stability in complex samples, reduce costs, and simplify operating procedures.⁶⁵

Summary and Prospects

CRAB remains one of the most serious antimicrobial resistance threats worldwide because of its diverse resistance mechanisms, rapid dissemination in healthcare settings, and limited therapeutic options. Over the past decade, substantial advances have been made in CRAB diagnostics, including phenotypic methods, molecular assays, sequencing technologies, mass spectrometry, biosensors, and AI-assisted approaches. However, no single diagnostic strategy currently achieves an optimal balance of speed, accuracy, and clinical applicability. Future development should therefore focus on improving diagnostic performance and generating information that better supports antimicrobial decision-making.⁶⁶

Current diagnostic approaches still have important limitations. Phenotypic antimicrobial susceptibility testing remains the reference standard but is limited by its long turnaround time. Molecular methods accelerate resistance detection, yet genotype-based prediction remains challenging because CRAB resistance results from multiple interacting mechanisms.⁶⁷ Future diagnostics should move beyond individual resistance markers toward more accurate prediction of antimicrobial susceptibility. A major challenge is that technological development has often outpaced clinical implementation. Many emerging platforms show excellent analytical performance under laboratory conditions, but their value in routine clinical practice remains uncertain.^68,69 Future studies should prioritize multicenter validation, standardized benchmarking, prospective implementation, and health-economic evaluation. Diagnostic technologies should be judged not only by analytical accuracy but also by their ability to improve treatment decisions and strengthen infection-control strategies.⁶⁹ Future progress will therefore depend not only on developing more advanced detection technologies but also on improving how diagnostic systems are designed, validated, and integrated into clinical workflows.⁷⁰

The future of CRAB diagnostics will be driven by clinical needs rather than by a single dominant technology. Over the next 5 years, rapid and decentralized platforms, particularly isothermal amplification, CRISPR-based assays, and biosensors, are expected to undergo broader clinical evaluation, especially for POCT. CRISPR-based assays and biosensor platforms appear particularly promising. They offer rapid detection and are increasingly suitable for decentralized testing. Within the next 10 years, nanopore sequencing and metagenomic sequencing are expected to play a greater role in resistance surveillance, outbreak investigation, and the diagnosis of complex infections. Integrated diagnostic platforms are also expected to become increasingly important. These platforms combine molecular detection, genomic surveillance, mass spectrometry, and AI-assisted analysis to provide more precise guidance for antimicrobial management. However, widespread adoption will require better validation, standardized workflows, and improved cost-effectiveness.⁷¹

Greater emphasis should now be placed on real-world evaluation rather than technological development alone. Multicenter clinical studies, standardized assessment frameworks, and prospective implementation studies will be essential for defining the practical value of emerging diagnostic platforms. Ultimately, the future of CRAB diagnostics will not be defined by a single breakthrough technology. Instead, progress will depend on a transition from technology-centered innovation to clinically grounded diagnostic design. This transition will be essential for developing rapid, reliable, and effective diagnostic strategies that improve antimicrobial stewardship, strengthen infection control, and help address the global challenge of antimicrobial resistance.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

Dr Qiuping Ye reports Support for the manuscript from Key Construction Discipline Scientific Research Ability Enhancement Project of Guangdong Province; Guangdong Provincial Key Laboratory of Functional Substances in Medicinal and Edible Plant Resources and Disease Prevention, during the conduct of the study. Mr Kang Zhang reports Support for the manuscript from Key Construction Discipline Scientific Research Ability Enhancement Project of Guangdong Province; Guangdong Provincial Key Laboratory of Functional Substances in Medicinal and Edible Plant Resources and Disease Prevention, during the conduct of the study. Mr Maoqing Tian reports Support for the manuscript from Key Construction Discipline Scientific Research Ability Enhancement Project of Guangdong Province; Guangdong Provincial Key Laboratory of Functional Substances in Medicinal and Edible Plant Resources and Disease Prevention, during the conduct of the study. Dr Zhenxia Zhang reports Support for the manuscript from Key Construction Discipline Scientific Research Ability Enhancement Project of Guangdong Province; Guangdong Provincial Key Laboratory of Functional Substances in Medicinal and Edible Plant Resources and Disease Prevention, during the conduct of the study. Dr Min Lin reports Support for the manuscript from Key Construction Discipline Scientific Research Ability Enhancement Project of Guangdong Province; Guangdong Provincial Key Laboratory of Functional Substances in Medicinal and Edible Plant Resources and Disease Prevention, during the conduct of the study. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.