Abstract
<title>Abstract</title> <p> Uropathogenic <italic>Escherichia coli</italic> (UPEC) are the leading cause of urinary tract infections (UTIs) and the virulence factor PapGII is linked to development of urosepsis. Current culture-based diagnostic assays are slow and do not include risk-assessment of the pathogen’s potential invasiveness. Therefore, clinicians rely on empirical antibiotic prescription choices, which contributes to evolution of antimicrobial resistance. We aimed to develop and validate a Loop Mediated Isothermal Amplification (LAMP)-based assay for the rapid detection of <italic>papGII</italic> -positive <italic>E. coli</italic> directly from urine. We designed specific primers targeting conserved regions of the <italic>E. coli uidA</italic> and <italic>papGII</italic> genes. To validate the assay, we collected 191 consecutive urine specimen samples over a three-day period from routine diagnostics. Culture results from routine diagnostics were used as reference for <italic>E. coli</italic> detection and whole genome sequencing for <italic>papGII</italic> detection. Our assay showed a limit of detection of 10 <sup>3</sup> CFU/ml, allowing detection at bacterial concentrations relevant for UTI. Among the clinical urine samples tested, the assay showed 87% sensitivity and 95% specificity for <italic>E. coli</italic> , and 100% sensitivity and specificity for <italic>papGII</italic> . Notably, all results were obtained within 40 minutes and without extensive sample preparation, highlighting the potential of early UPEC identification with an increased risk of invasiveness. </p>