Simplified bacterial identification and antimicrobial resistance detection in bacteraemia by on-chip multiplexed LAMP...

Bacteraemia is a severe infection associated with high risks of complications and mortality. Diagnosis relies mainly on blood cultures, followed by culture-based methods, which remain the gold-standard but are time-consuming. Isothermal bacterial DNA amplification provides a rapid, reliable and cost-effective alternative for identifying and characterising bacteria directly from positive blood cultures, enabling a timelier diagnosis, particularly in resources-limited settings. However, routine implementation has been hindered by sample processing requirements and its limited multiplexing capacity. Here, we developed an on-chip loop-mediated isothermal amplification (LAMP) workflow, ALADIM (accessible and integrated LAMP analysis for multiplex detection of infectious agents in microfluidics), that integrates bacterial extraction and multiplex LAMP, followed by instrument-free amplicons detection using lateral flow immunoassay (LFIA), all within two hours (vs. 24-48 hours for the gold-standard). To demonstrate the capabilities of our device, we targeted the most common bacteria found in bacteraemia, E. coli, the co-amplification of two highly prevalent antimicrobial resistance genes, bla_CTX-M-15 and bla_OXA-48, and the detection of a positive control. Multiplex LAMP followed by LFIA detection was first evaluated using 14 strains cultured in LB. On-chip results were comparable to those obtained with our reference method (tube-based LAMP in a thermocycler followed by LFIA detection), showing 100% sensitivity, >84% specificity for the detection of malF and bla_OXA-48, and 68% sensitivity, 83% specificity for bla_CTX-M-15. As a proof of concept for the applicability of the complete ALADIM workflow, we analysed positive overnight blood cultures spiked with 15 strains resulting in 61 independent experiments (DNA extraction/amplification/LFA readout). In this complex matrix, ALADIM specificity remained above 77% for all targets, while sensitivity exceeded 94% for malF and bla_OXA-48 but decreased to 56% for bla_CTX-M-15. A similar decrease in overall sensitivity was observed for the in-tube method (>68%), highlighting the challenges associated with direct whole-blood analysis. This feasibility study shows the integration of multiplex DNA amplification, microfluidic processing, and LFIA detection in a single platform. Future work will focus on improving sample preparation, assay performance in complex matrices, and expanding multiplexing capabilities of ALADIM towards additional targets, to deliver a clinically reliable point-of-care testing device for low- and middle-income countries.