Development and Evaluation of a Handmade Lateral Flow Immunoassay for the Rapid Detection of Carbapenem-producing Gram-Negative Bacteria in China

Cai-Yun Zhang,1,* Jun Ji,2,* Xiu-Zhen Chen,3,4 Wei-Xiao Wang,3,4 Wei Chen,3,4 Xiao-Li Cao2 1The key Laboratory of Hepatology of Nanjing, the Second Hospital of Nanjing, Affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210003, People’s Republic of China; 2Department of Clinical Laboratory, Nanjing Drum Tower Hospital, Affiliated to Nanjing University, Nanjing, Jiangsu, 210008, People’s Republic of China; 3Department of Tuberculosis, the Second Hospital of Nanjing, Affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210003, People’s Republic of China; 4Clinical Research Center, the Second Hospital of Nanjing, Affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210003, People’s Republic of China*These authors contributed equally to this workCorrespondence: Wei Chen, Email [email protected] Xiao-Li Cao, Email [email protected]: The global dissemination of carbapenemase-producing organisms (CPOs) poses a critical public health threat. Rapid, cost-effective diagnostic tools are urgently needed to guide antimicrobial therapy and mitigate resistance spread.Objective: This study aimed to develop and evaluate a lateral flow immunoassay (LFIA)-based workflow for the rapid detection of carbapenemases (KPC, IMP, NDM, VIM, and OXA-48) in clinical settings.Materials and Methods: The LFIA strips targeting carbapenemases were handmade in-house using monoclonal antibodies specific to five prevalent subtypes: KPC, IMP, NDM, VIM, and OXA-48. Performance was validated using 26 characterized carbapenemase-producing Enterobacterales (CPE) strains (10 KPC, 5 IMP, 7 NDM, 1 VIM, and 3 OXA-48 producers) and 105 clinical isolates. Among them, strains positive for carbapenemase by LFIA were further confirmed by Next-generation sequencing (NGS) and PCR, and their carbapenem susceptibility was determined by the Vitek2 Antibiotics Susceptibility Test (AST). Diagnostic metrics were further assessed through limit of detection (LoD), cross-reactivity, and interference studies. The overall workflow for the development and evaluation of the LFIA is presented in Figure 1.Results: The LFIA demonstrated 100% sensitivity in detecting all 26 CPE strains. Among 105 clinical isolates, 44 were identified as CPO strains, with strong concordance to PCR/NGS results (discrepancies in 8 isolates observed between PCR and NGS). The assay exhibited high sensitivity (100%), specificity (100%), positive predictive value (PPV, 93.2%), negative predictive value (NPV, 100%), and accuracy (97.1%). The LoD for each carbapenemase type was established. No cross-reactivity with non-target bacteria or interference from clinical substances was observed. The workflow achieved results in 15 minutes at a lower cost than conventional methods.Conclusion: This LFIA-based workflow provides a rapid, reliable, and cost-effective solution for detecting CPOs, particularly for critically ill patients or in resource-limited settings. Its implementation could enhance therapeutic decision-making and antimicrobial stewardship.Keywords: carbapenemase-producing organisms, lateral flow immunoassay, carbapenem-resistance, rapid detection, cost-effective workflow