Infectious diseases remain a leading cause of death globally, exacerbated by the rise of antibiotic resistance. Gram-negative pathogens, especially carbapenemase-producing Enterobacteriaceae (CPE), are a significant concern due to their increasing prevalence and resistance to multiple antibiotics. CPE produce carbapenemase, an enzyme that inactivates carbapenems, a last-line antibiotic. Conventional detection methods, like the modified Hodge test and mCIM, are time-consuming and may lack optimal accuracy. Molecular methods like PCR are costly and resource-intensive. The Carba NP test offers a rapid chromogenic assay, but it involves manual steps. This research addresses the need for a miniaturized, rapid, accurate, and parallelized CPE detection platform by utilizing magnetic digital microfluidics (MDM) technology. MDM offers advantages in handling small fluid volumes, ease of automation, and potential for point-of-care (POC) applications in resource-limited settings. This paper details the development of MDM Carba, a platform integrating the Carba NP assay with MDM technology for efficient and rapid CPE detection.

The literature extensively documents the global threat of antibiotic resistance, particularly from Gram-negative bacteria like CPE. Existing methods for CPE detection, including phenotypic assays (modified Hodge test, mCIM) and molecular assays (PCR), have limitations in speed, accuracy, and resource requirements. The Carba NP test, while faster than traditional methods, still requires manual liquid handling steps. The authors highlight the advantages of MDM technology over other microfluidic approaches, emphasizing its suitability for biochemical reactions and POC diagnostics due to simplified fluidic manipulation and the dual functionality of magnetic particles. Prior work demonstrates successful MDM applications in molecular testing, immunotesting, and antimicrobial susceptibility testing, showcasing its potential for in vitro diagnostic platforms.

MDM Carba utilizes the Carba NP assay, measuring pH changes resulting from carbapenem hydrolysis by carbapenemase. The platform consists of a 3D-printed droplet manipulator, a Teflon-modified glass substrate, and a magnet array. Twelve parallel reactions are possible. The device manipulates droplets containing bacterial lysate, magnetic particles, and reaction buffer (with or without imipenem) using magnetic force. The color change of the droplets (red to yellow/orange) indicates CPE presence. A manual version and a motorized automated version were developed. The automated system incorporates a step motor, potentiometer, and Bluetooth/USB connectivity for smartphone and PC control. The process involves loading reagents, adding bacterial samples, droplet manipulation (mixing and merging), incubation, and colorimetric readout. The study validated MDM Carba by testing 27 bacterial isolates, comparing results with molecular testing and conventional Carba NP. Droplet volume, mixing efficiency and homogeneity were optimized. Control reactions without imipenem were performed for validation.

The MDM Carba platform successfully detected CPE with a turnaround time of approximately 125 minutes (reducible to 65 minutes by shortening incubation). The device demonstrated efficient droplet manipulation and mixing using a 3D-printed micropillar-based passive mixer. The sensitivity and specificity of MDM Carba were comparable to the Carba NP test. The automated version provided consistent and reliable performance. The platform successfully tested six bacterial strains in parallel reactions using manual and automated methods. The use of magnetic particles facilitated efficient mixing, and the polydopamine coated hydrophilic droplet holders enabled immobilization and easy observation of the colorimetric changes. The study demonstrated the feasibility of a 3D-printed MDM device for rapid and efficient CPE detection, suggesting potential for point-of-care diagnostics. Reagent consumption was reduced by up to 90% compared to conventional methods. The device has a modular design that can adapt to other antibiotic resistance tests.

MDM Carba addresses the limitations of existing CPE detection methods by providing a rapid, accurate, and cost-effective solution. The comparable sensitivity and specificity to the Carba NP test, along with reduced reagent consumption and potential for automation and POC application, highlight its significant advantages. The platform's modular design facilitates adaptation for detecting other antibiotic resistance mechanisms. The successful integration of 3D printing and MDM technology opens new avenues for developing portable and affordable diagnostic tools in resource-constrained settings. Further research could focus on optimizing the device design, expanding its testing capabilities, and conducting larger clinical trials to assess its performance in real-world settings.

MDM Carba represents a significant advance in rapid CPE detection, combining the advantages of the Carba NP assay with the efficiency and portability of MDM technology. The platform's performance, ease of use, and potential for point-of-care applications make it a promising tool for improving clinical management of CPE infections. Future work should focus on expanding the platform's capabilities to detect other resistance mechanisms and conducting larger-scale clinical validation studies.

The study involved a relatively small number of bacterial isolates. Further validation with a larger and more diverse set of isolates is necessary. The current design may need further optimization to fully automate all steps of the process. Long-term stability and durability of the 3D-printed components need to be assessed. The study primarily focused on laboratory performance; evaluation in real-world clinical settings is essential.