Culture-Free Rapid Phenotypic Antimicrobial Susceptibility Testing for Helicobacter pylori Based on Fluorescence Rapid On-Site Evaluation Technology: A Preliminary Study

Abstract

BackgroundPhenotypic 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. MethodsWe 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 {degrees}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 Cohens kappa coefficient were calculated. ResultsFourteen 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. Cohens kappa coefficients indicated moderate agreement for all three antibiotics ({kappa} = 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. ConclusionsThis 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.