Background: Phenotypic antibiotic susceptibility testing (AST) for Helicobacter pylori (H. pylori) has relied on bacterial culture for three decades, requiring 5–7 days to yield results. Genotypic rapid tests can only detect known resistance mutations and fail to reliably identify amoxicillin resistance. To our knowledge, no culture-free rapid phenotypic AST method for H. pylori has been previously reported. Methods: We developed a phenotypic AST method based on fluorescence rapid on-site evaluation (ROSE) technology that completely bypasses bacterial culture. Gastric mucosal biopsy specimens from 40 H. pylori-positive patients were homogenized and co-incubated with an acridine orange/ethidium bromide (AO/EB)-based viability staining reagent and three first-line antibiotics—amoxicillin, clarithromycin, and levofloxacin—at concentrations corresponding to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for H. pylori, at 37 °C for 1 hour. Fluorescence intensity was measured using a microplate reader. A reduction in fluorescence relative to an antibiotic-free control indicated susceptibility, whereas no significant reduction indicated resistance. Conventional culture-based AST (E-test) served as the reference method. The overall concordance rate, sensitivity, specificity, and Cohen’s kappa coefficient were calculated. Results: Fourteen of the 40 samples had unsuccessful culture and were excluded, leaving 26 samples for statistical analysis of each antibiotic. The overall concordance rates between the ROSE method and culture-based AST were 84.6% (22/26) for amoxicillin, 76.9% (20/26) for levofloxacin, and 69.2% (18/26) for clarithromycin. Cohen’s kappa coefficients indicated moderate agreement for all three antibiotics (κ = 0.523, 0.539, and 0.412, respectively). Unlike genotypic methods, the ROSE method successfully assessed amoxicillin susceptibility in all 40 patients—a critical first-line antibiotic for which no reliable genetic resistance marker currently exists. The turnaround time was approximately 1 hour (55–65 minutes), compared with 5–7 days for culture-based methods; preliminary estimates indicated a cost reduction of approximately 3,000–5,000 Chinese yuan (CNY) per patient, mainly attributable to the elimination of culture media, prolonged incubation, and repeat clinic visits. Conclusions: This study reports, for the first time, a culture-free 1-hour phenotypic AST for H. pylori. The method enables same-day, susceptibility-guided treatment decisions, addressing an unmet clinical need spanning three decades. Algorithm optimization and a prospective randomized controlled trial are currently underway to further improve diagnostic accuracy and validate clinical utility.

Objective: This study was designed to evaluate the diagnostic performance of fluorescence-based rapid on-site specimen evaluation (F-ROSE) for antimicrobial susceptibility testing of Helicobacter pylori ( H. pylori ) and to compare test performance before and after iterative optimization of fluorescence image acquisition and of the artificial intelligence (AI) recognition algorithm, using two consecutive rounds of paired testing, so as to provide trial data supporting clinical implementation of rapid susceptibility testing. Methods: Forty patients with a urea breath test (UBT) or rapid urease test (RUT) positive for H. pylori within the preceding 2 weeks, together with strongly positive endoscopic findings, were prospectively enrolled. In order of enrollment they were allocated to two rounds in which F-ROSE was compared with culture-based susceptibility testing. Twenty patients were tested in round 1 on an automated fluorescence immunoassay scanner running the original algorithm; a further 20 were tested in round 2 with the optimized algorithm. With in vitro culture and E-test as the reference standard, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of F-ROSE were calculated for each round for amoxicillin, levofloxacin hydrochloride and clarithromycin, and agreement between methods was assessed with Cohen’s kappa. Receiver operating characteristic (ROC) analysis was used to identify the optimal cutoff of the fluorescence residual rate (FRR) and to determine how far threshold adjustment improved performance. Results: Culture succeeded in 15 patients in round 1 and in 14 patients in round 2, leaving 29 evaluable samples. Overall diagnostic performance after algorithm optimization was clearly better than before. Pooled across the three antibiotics, sensitivity was 92.9%, specificity 71.0% and accuracy 77.8% before optimization; after optimization sensitivity rose to 100.0%, specificity increased slightly to 74.2% and accuracy to 81.0%. NPV reached 100.0% for all three agents after optimization, and no resistant isolate was missed. The gain was largest for clarithromycin, for which sensitivity rose from 85.7% to 100.0%, accuracy from 66.7% to 78.6% and kappa from 0.348 to 0.571, indicating a clear improvement in agreement between the two methods. ROC analysis showed that drug-specific optimal cutoffs derived from the Youden index improved specificity appreciably compared with the uniform 50% FRR threshold currently applied, the gain being most evident for levofloxacin hydrochloride. Conclusions: With sharper fluorescence images and an improved AI recognition algorithm, F-ROSE performs better in H. pylori susceptibility testing, and the reduction in missed resistant strains is particularly noteworthy; the assay is therefore a plausible option for rapid susceptibility screening in clinical practice. Multicenter studies with larger samples are still required to confirm the stability of the technique.

Objective: To establish a standardized training program for endoscopic pathogen visualization literacy (EPVL) based on fluorescence rapid on-site evaluation (ROSE) technology for gastroenterologists, and to evaluate its training efficacy. Methods: A prospective quasi-experimental study was conducted. A total of 54 gastroenterology trainees were non-randomly allocated into the EPVL training group (Group A, n=28, 16-hour comprehensive training) and the control group (Group B, n=26, 3.5-hour traditional teaching). Pre- and post-training assessments included theoretical examinations, fluorescence ROSE image interpretation tests (30 parallel images per set), interpretation speed measurement, and clinical decision-making integration evaluation. The primary outcome was the change in image interpretation accuracy, analyzed by ANCOVA with pre-test scores as the covariate. Results: Baseline characteristics were comparable between groups (P>0.05 for all demographic variables and pre-test scores). Group A showed significant improvement in image interpretation accuracy from 57.8±13.6% pre-training to 82.5±11.2% post-training (improvement of 24.7%, paired t=-12.86, P<0.001), while Group B improved from 58.5±13.0% to 71.0±13.5% (improvement of 12.5%, paired t=-5.24, P<0.001). After ANCOVA adjustment for pre-test scores, the between-group difference was significant (F(1, 51)=10.95, P=0.0017, η ² =0.177), with Cohen’s d=0.94 (large effect size). Interpretation speed in Group A (19.2±2.8 s/image) was significantly faster than in Group B (32.5±6.0 s/image, t=-10.45, P<0.001). Clinical decision-making scores were significantly higher in Group A (80.5±8.0 vs. 65.3±11.5, t=5.60, P<0.001). The Kappa agreement with the gold standard in Group A improved from 0.56±0.18 to 0.84±0.11 (t=-8.35, P<0.001). Participant satisfaction exceeded 88%. Conclusion: The EPVL training program significantly improves gastroenterologists’ fluorescence ROSE image interpretation accuracy, speed, and clinical decision-making integration, providing a novel and effective standardized training paradigm for digestive endoscopy education.

Background Phenotypic antibiotic susceptibility testing (AST) for Helicobacter pylori (H. pylori) has relied on bacterial culture for three decades, requiring 5–7 days to yield results. Genotypic rapid tests can only detect known resistance mutations and fail to reliably identify amoxicillin resistance. To our knowledge, no culture-free rapid phenotypic AST method for H. pylori has been previously reported. Methods We developed a phenotypic AST method based on fluorescence rapid on-site evaluation (ROSE) technology that completely bypasses bacterial culture. Gastric mucosal biopsy specimens from 40 H. pylori-positive patients were homogenized and co-incubated with an acridine orange/ethidium bromide (AO/EB)-based viability staining reagent and three first-line antibiotics—amoxicillin, clarithromycin, and levofloxacin—at concentrations corresponding to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for H. pylori, at 37°C for 1 hour. Fluorescence intensity was measured using a microplate reader. A reduction in fluorescence relative to an antibiotic-free control indicated susceptibility, whereas no significant reduction indicated resistance. Conventional culture-based AST (E-test) served as the reference method. The overall concordance rate, sensitivity, specificity, and Cohen's kappa coefficient were calculated. Results Fourteen of the 40 samples had unsuccessful culture and were excluded, leaving 26 samples for statistical analysis of each antibiotic. The overall concordance rates between the ROSE method and culture-based AST were 84.6% (22/26) for amoxicillin, 76.9% (20/26) for levofloxacin, and 69.2% (18/26) for clarithromycin. Cohen's kappa coefficients indicated moderate agreement for all three antibiotics (κ = 0.523, 0.539, and 0.412, respectively). Unlike genotypic methods, the ROSE method successfully assessed amoxicillin susceptibility in all 40 patients—a critical first-line antibiotic for which no reliable genetic resistance marker currently exists. The turnaround time was approximately 1 hour (55–65 minutes), compared with 5–7 days for culture-based methods; preliminary estimates indicated a cost reduction of approximately 3,000–5,000 Chinese yuan (CNY) per patient, mainly attributable to the elimination of culture media, prolonged incubation, and repeat clinic visits. Conclusions This study reports, for the first time, a culture-free 1-hour phenotypic AST for H. pylori. The method enables same-day, susceptibility-guided treatment decisions, addressing an unmet clinical need spanning three decades. Algorithm optimization and a prospective randomized controlled trial are currently underway to further improve diagnostic accuracy and validate clinical utility.