New Study Compares MALDI-TOF MS Assays for Detecting Carbapenemase Activity

Carbapenem is a class of antibiotics that has increasingly been used to treat infections from bacteria that are resistant to other commonly used antibiotics. Carbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE), driven by their resistance to carbapenem antibiotics, now pose a significant global health threat.

In a recent study published in Scientific Reports¸ Austrian researchers from the Medical University of Graz compared two matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS)-based assays that have routinely been used to detect carbapenemase activity and identify CRE.

By evaluating their sensitivity, specificity, and ease of use, the study aimed to determine the reliability and practicality of each assay in the diagnostic setting for effective CRE management.

Antibiotic resistance​​​​​​​Study: A comparison of two MALDI-TOF MS based assays for the detection of carbapenemases in Enterobacterales. Image Credit: ART-ur/Shutterstock.com

Background

The development of antimicrobial resistance poses a major health concern, increasing the risk of nosocomial infections. The emergence of carbapenem-resistant Gram-negative bacteria has been especially concerning since carbapenem has hitherto been used to treat bacterial infections that did not respond to other commonly used antibiotics.

Carbapenemase enzymes that confer the ability to break down carbapenem antibiotics are primarily responsible for carbapenem resistance in Enterobacterales. Early and accurate detection of carbapenemases is crucial for guiding appropriate treatment, limiting the spread of resistance, and improving patient outcomes.

While molecular and immunologic tests exist for carbapenemase detection, they have limitations in scope and enzyme coverage or require specialized handling. However, MALDI-TOF MS offers a promising approach by detecting carbapenem hydrolysis products, irrespective of the enzyme type.

Until recently, Bruker’s MBT STAR-Carba IVD Kit was the sole commercial MALDI-TOF assay available for detecting carbapenemase activity. However, the Zybio Carbapenemase Activity Kit is now emerging as a potential alternative.

About the Study

In the present study, the scientists aimed to understand the capabilities and limitations of the two assays commercially available for detecting carbapenemase activity, which is essential for determining their role in routine diagnostics and managing the carbapenem resistance crisis.

They evaluated the two MALDI-TOF MS-based assays — the MBT STAR-Carba IVD Kit by Bruker and the Carbapenemase Activity Kit by Zybio — for detecting carbapenemase in CRE isolates. A total of 40 CRE samples were pre-selected based on carbapenem resistance, identified through routine laboratory methods, and used to test the performance of these assays.

The study employed Bruker's MALDI Biotyper and Zybio's EXS2600 for sample analysis. Each assay followed manufacturer protocols, with the Bruker assay using automated spectrum acquisition and the Zybio assay being tested initially through the automated protocol but also through manual spot targeting, as recommended for enhanced accuracy.

For quality control, positive and negative controls, including known carbapenemase-producing and non-carbapenemase-producing isolates, respectively, of Klebsiella pneumoniae were tested alongside clinical samples. The results were interpreted based on the detection of hydrolyzed carbapenems, which were categorized as positive for carbapenemase.

The analytical part of the study focused on obtaining sensitivity, specificity, and predictive values for each assay to assess diagnostic accuracy.

Furthermore, the protocols used in the study adhered to clinical diagnostic standards, ensuring that the findings on the reliability and reproducibility of the assays could inform their routine diagnostic application in healthcare settings.

Major findings

The study found that both assays demonstrated high sensitivity in detecting carbapenemase-producing Enterobacterales but varied in their specificity and user handling requirements.

The Bruker assay required duplicate testing and the test reagents needed to be used immediately post-opening, whereas the Zybio assay could be used within a week under refrigerated storage.

Furthermore, the targets in the Bruker assay were single-use, while the Zybio assay allowed their steel targets to be reused after a quick cleaning protocol.

Additionally, the calibration and software requirements also differed, with the Bruker assay’s system being fully automated in sample processing and measurement, simplifying operational efficiency but limiting user flexibility.

In contrast, the Zybio assay provided adjustable settings for optimizing measurements when high background noise was encountered, allowing repeat analysis from the same sample target without re-preparation.

Notably, Bruker's MBT STAR-Carba IVD Kit achieved a sensitivity of 92% and a specificity of 91%, accurately identifying carbapenemase in most cases with a lower incidence of false positives. Among the tested isolates, three yielded unclear results in Bruker’s assay, which required retesting. However, 93% of the samples produced valid results on the first attempt.

The Zybio Carbapenemase Activity Kit showed a slightly higher sensitivity at 96%, but the specificity was lower at 64%, with more false positives.

Automatic spectrum acquisition on Zybio’s system yielded fewer valid results initially (53%) than the manual analysis (85%), indicating the benefit of manual intervention.

Moreover, Zybio’s assay had four false-positive cases, primarily in isolates without confirmed carbapenemase genes. The positive predictive values were 96% for the Bruker assay and 85% for the Zybio assay, while the negative predictive values were 83% and 88%, respectively.

Additional handling differences were also observed, with Bruker’s automated software simplifying usage but limiting user adjustments, while the Zybio assay allowed flexibility in settings, potentially reducing inconclusive results.

However, the researchers believe that although Zybio’s reusable target plates provided cost benefits, they might contribute to specificity challenges due to cross-sample interference.

Conclusions

The study concluded that both MALDI-TOF MS assays provide reliable options for carbapenemase detection in routine diagnostics.

The Bruker MBT STAR-Carba IVD Kit showed superior specificity, while Zybio’s Carbapenemase Activity Kit offered flexibility and higher sensitivity. The researchers recommended that a choice between these systems should be based on laboratory needs, such as accuracy priorities, operational simplicity, and cost efficiency.

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