Standardized quantitative imaging of extracellular polymeric substance in chronic wounds: Prognostic comparison of wound blotting and bacterial fluorescence imaging

Bacterial burden and biofilm-associated wound pathology are important contributors to delayed wound healing. However, conventional diagnostic methods are time-consuming and do not provide real-time, objective quantification of bacterial fluorescence or biofilm-associated wound-bed matrix signals, limiting their value for timely clinical decision-making. Bedside technologies such as MolecuLight i:X and the Wound Biofilm Detection Kit (WBDK) enable rapid visualization of bacterial fluorescence and Alcian blue–positive wound-blotting signals, respectively, but their interpretation largely depends on subjective visual assessment. This retrospective cohort study developed a standardized image-analysis approach to objectively quantify bacterial fluorescence signals detected by MolecuLight i:X and Alcian blue–positive wound-blotting matrix signals detected by WBDK. Clinical images from 103 chronic wound cases were retrospectively analyzed. Image-processing protocols were established to quantify fluorescence intensity as a surrogate marker of bacterial burden and WBDK staining intensity as a quantitative measure of biofilm-associated matrix signals. These quantitative metrics were evaluated for their associations with 90-day wound healing outcomes. WBDK intensity demonstrated strong discriminative ability for predicting 90-day wound healing (AUC = 0.921, 95% CI 0.878–0.975). In contrast, MolecuLight i:X fluorescence intensity showed moderate predictive performance (AUC = 0.732, 95% CI 0.625–0.839). Per one–standard deviation increase, the adjusted risk ratio for non-healing was 2.05 for WBDK intensity and 1.41 for fluorescence intensity. Objective quantification of WBDK-derived biofilm-associated matrix signals provides stronger prognostic information than bacterial fluorescence intensity alone and may enable more reproducible stratification of chronic wounds.