is a leading cause of healthcare-associated infections worldwide. Currently, there is a lack of consensus for an optimal diagnostic method for infection (CDI). Multi-step diagnostic algorithms use enzyme immunosorbent analysis (EIA)-based detection of toxins TcdA/TcdB in stool, premised on the rationale that EIA toxin-negative (Tox) patients have less severe disease and shorter diarrhoea duration. The aim of this study was to characterize toxigenic (i.e. -positive) strains isolated from diarrheic patient stool with an EIA Tox (i.e. "discrepant") CDI diagnostic test result. Recovered strains were DNA fingerprinted (ribotyped), subjected to multiple toxin, genome and proteome evaluations, and assessed for virulence. Overall, of 1243 -positive patient stool specimens from Southern Arizona hospitals, 31% were discrepant. For RT027 (the most prevalent ribotype)-containing specimens, 34% were discrepant; the corresponding RT027 isolates were cytotoxic to cultured fibroblasts, but their total toxin levels were comparable to, or lower than, the historic low-toxin-producing strain CD630. Nevertheless, these low-toxin RT027 strains (LT-027) exhibited similar lethality to a clade-matched high-toxin RT027 strain in Golden Syrian hamsters, and heightened colonization and persistence in mice. Genomics and proteomics analyses of LT-027 strains identified unique genes and altered protein abundances, respectively, relative to high-toxin RT027 strains. Collectively, our data highlight the robust virulence of LT-027 , provide a strong argument for reconsidering the clinical significance of a Tox EIA result, and underscore the potential limitations of current diagnostic protocols.