Clinical comparative study of fluorescence rapid on-site evaluation (F-ROSE) real-time visual detection versus ¹³C-urea breath test for helicobacter pylori.

BACKGROUND: ¹³C-urea breath test (¹³C-UBT) is widely used for Helicobacter pylori (H.pylori) detection but has limitations including inability to distinguish viable from nonviable bacteria, an ambiguous diagnostic gray zone (DOB 4-10), and false-negative results in low bacterial load infections. OBJECTIVES: To compare the diagnostic value of fluorescence rapid on-site evaluation (F-ROSE) and ¹³C-UBT for H. pylori infection, determine the critical viable bacterial count threshold for positive ¹³C-UBT, clarify the real infection status in the ¹³C-UBT gray zone, and reveal the quantitative relationship between DOB values and viable bacterial counts. METHODS: A total of 180 patients undergoing gastroscopy were consecutively enrolled and received synchronous F-ROSE quantitative detection and ¹³C-UBT. F-ROSE used acridine orange-ethidium bromide (AO-EB) dual fluorescent staining to count total, viable, and nonviable bacteria under × 400 high-power field (HPF). ¹³C-UBT results were determined by DOB values. Latent class analysis (LCA) combined with Bayesian modeling was used to infer true infection status. Receiver operating characteristic (ROC) curves were applied to define the critical viable bacterial count for positive ¹³C-UBT and evaluate diagnostic efficacy. RESULTS: Model inference identified 94 true-positive and 86 true-negative cases among 180 patients. The positive rate of F-ROSE was 51.1%, significantly higher than 47.2% of ¹³C-UBT (P < 0.05). Using true infection as reference: F-ROSE showed sensitivity of 93.6%, specificity of 94.2%, and AUC of 0.954; ¹³C-UBT showed sensitivity of 80.9%, specificity of 93.0%, and AUC of 0.878. ROC analysis indicated that a viable bacterial count≥10/HPF was the stable positive threshold for ¹³C-UBT. When viable bacterial count was <10/HPF, the positive rate of ¹³C-UBT was only approximately 20%, with most cases falling into the gray zone or false-negative. There were 32 cases in the ¹³C-UBT gray zone, among which 24 were positive by F-ROSE (positive rate 75.0%), all representing true infections with low viable bacterial load (4-9/HPF). The ¹³C-UBT DOB value was significantly positively correlated with F-ROSE viable bacterial count (r = 0.746, P < 0.001). CONCLUSIONS: ¹³C-UBT positivity depends on a viable bacterial count threshold ≥10/HPF. Infections with low viable bacterial count are prone to be classified into the gray zone or missed. The clinical gray zone mostly represents true viable bacterial infection, and the traditional strategy of "observation first" is biased. F-ROSE enables real-time visual identification of viable bacteria and accurate quantification, breaking through the threshold limitation of ¹³C-UBT, making it an optimal technique for accurate diagnosis of H. pylori infections with low bacterial load and in the clinical gray zone.