Development and Evaluation of a Duplex Lateral Flow Immunoassay for Rapid Detection of KPC and NDM Carbapenemases

Background: Carbapenem-resistant Enterobacterales represent a critical global health challenge, with KPC and NDM being the most prevalent carbapenemase families. Rapid and reliable detection tools are essential for guiding infection control and antimicrobial stewardship, particularly in resource-limited settings. Methods: A lateral flow immunoassay (LFIA) strip was developed for simultaneous detection of KPC and NDM carbapenemases. Monoclonal antibody pairs were screened and assay parameters were optimized to improve analytical performance. Analytical evaluation was performed using recombinant proteins. Clinical evaluation was conducted using 12 non-duplicate clinical Klebsiella pneumoniae ( K. pneumoniae ) isolates, with bla KPC / bla NDM confirmed by PCR and whole-genome sequencing (WGS). Results: The LFIA achieved limits of detection of 0.5 ng/mL for KPC and 0.1 ng/mL for NDM using recombinant proteins. No cross-reactivity was observed with other carbapenemases (OXA-48, VIM, IMP). In the clinical K. pneumoniae set, LFIA results showed 100% concordance with the PCR/WGS reference. Test results were visually interpretable within 15 minutes without specialized instrumentation. Conclusion: This duplex LFIA provides rapid, instrument-free visual screening for KPC and NDM carbapenemases in cultured isolates, supporting routine laboratory screening and workflow decisions. Larger multi-center studies across diverse Enterobacterales species and carbapenemase variants will further define its broader clinical performance. Keywords: lateral flow immunoassay, KPC, NDM, carbapenem-resistant Enterobacterales