One-Hour Culture-Free Phenotypic Antimicrobial Susceptibility Testing for Helicobacter pylori Based on Fluorescence Metabolic Activity: A Preliminary Validation Study

Corresponding author: Zhanglin, stepinghuns2@163.com

DOI: 10.12201/bmr.202607.00011

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  Abstract: Background:The escalating rate of clinical antimicrobial resistance in Helicobacter pylori (H.pylori) has underscored the urgent need for susceptibility-guided individualized eradication strategies. Conventional culture-based antimicrobial susceptibility testing (AST) faithfully reflects the resistance phenotype of clinical strains but is hindered by prolonged turnaround times, while molecular detection methods offer rapid results yet are restricted to known resistance determinants and cannot identify novel mutations. To address these limitations, the present study aimed to establish and validate a novel culture-free, point-of-care phenotypic AST platform for H. pylori.Methods:Forty patients with confirmed H. pylori infection were enrolled. Gastric antrum mucosal biopsy specimens were mechanically homogenized to prepare bacterial suspensions, which were then subjected to susceptibility testing for amoxicillin, clarithromycin, and levofloxacin at concentrations based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints. Viable and non-viable bacteria were differentially labeled using dual fluorescent probes, with antibiotic-free suspensions serving as blank controls. Following incubation at 37°C for 60 minutes, fluorescence emission intensities were quantified by microplate reader, and susceptibility or resistance was determined by comparing fluorescence changes relative to the blank control. The conventional culture-based E-test method served as the reference standard. Sensitivity, specificity, Cohen's kappa coefficient, and concordance rates with E-test were calculated to evaluate the diagnostic performance of the Fluorescence Rapid On-Site Evaluation (F-ROSE) method. Results:H.pylori was detected in all 40 specimens by F-ROSE, and susceptibility profiles for all three antibiotics were simultaneously determined. Fourteen specimens were subsequently excluded due to unsatisfactory growth on culture media, leaving 26 specimens per antibiotic available for performance evaluation. The overall concordance rates between F-ROSE and E-test were 84.6% (22/26) for amoxicillin, 76.9% (20/26) for levofloxacin, and 69.2% (18/26) for clarithromycin. Cohen's kappa coefficients were 0.523 (95% CI: 0.093–0.953), 0.539 (0.215–0.862), and 0.412 (0.074–0.751) for the three agents, respectively, all indicating moderate agreement.Conclusions:This study presents the first culture-free rapid phenotypic AST system (F-ROSE) for H. pylori, capable of delivering susceptibility results within approximately one hour and thereby overcoming the longstanding bottleneck of conventional culture-based approaches that require several days. This platform holds promise for enabling real-time clinical decision-making and guiding individualized targeted eradication regimens, potentially resolving the clinical dilemma in which empirical therapy must be initiated before susceptibility data become available. Nevertheless, the limited sample size of this pilot study warrants further large-scale prospective investigations to optimize assay parameters, and randomized controlled trials are needed to validate the impact of this technology on H. pylori eradication outcomes and to support its broader clinical implementation.

  Key words: Helicobacter pylori; antimicrobial susceptibility testing; phenotypic susceptibility; culture-free; fluorescence rapid on-site evaluation; rapid diagnosis

  Submit time: 26 August 2026

  Copyright: The copyright holder for this preprint is the author/funder, who has granted biomedRxiv a license to display the preprint in perpetuity.
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  1	2026-07-05	10.12201/bmr.202607.00011V1	Download

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