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Objective To assess the usefulness of contrast-enhanced ultrasound (CEUS) in the differential diagnosis between malignant and benign gallbladder (GB) diseases.Methods This study had institutional review board approval, and informed consent was obtained from all patients.A total of 192 patients from 9 university hospitals were studied;lesions included GB adenocarcinoma (n=51), cholesterol polyp (n=63), adenomas (n=21), adenomyomatosis (n=14), chronic cholecystitis (n=31), and biliary sludge (n=12).After intravenous bonus injection of a contrast agent of phospholipid-stabilized microbubbles filled with sulfur hexafluoride, lesions were scanned with low acoustic power CEUS in the arterial and venous phases.A multiple logistic regression analysis was used to identify diagnostic features from 17 independent variables that enabled differentiation between malignant and benign GB diseases.The independent variables with P values of less than 0.05 in the multiple logistic regression analysis were selected for receiver operating characteristic (ROC) curve analysis.Results Among the 17 independent variables, multiple logistic regression analysis showed that the following 8 independent variables were associated with the nature of the GB diseases, including the patient age, GB wall thickness, lesion location, arrival time to the lesion, time-to-peak, washout time, intralesional blood vessels during arterial phase, and wall intactness (all P<0.05).ROC analysis showed that the intralesional blood vessels during the arterial phase of CEUS and the intactness of the GB wall depicted on CEUS were the most significant independent variables.The corresponding area under the ROC curve, sensitivity, specificity, and Youdens index for the intralesional blood vessels were 0.831 (95% confidence interval: 0.784~0.879), 63.6%, 98.0%, and 0.616, respectively, and for the GB wall intactness were 0.854 (95% confidence interval: 0.785~0.923), 78.4%, 92.9%, and 0.713, respectively.Conclusion CEUS is a useful tool in differentiating malignant from benign GB diseases.The branched tumor vessels and destruction of GB wall on CEUS are highly suggestive of GB malignancy.