通讯作者: 张林, stepinghuns2@163.com

DOI：10.12201/bmr.202607.00011

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

• 摘要：背景：随着幽门螺杆菌临床耐药率逐年升高，依托药敏指导下的个体化精准根除需求日益迫切。传统培养法药敏实验可真实反映菌株耐药表型，但培养周期冗长；分子检测速度快，仅能检出已知耐药靶点，无法识别未知突变。针对两类检测方法的缺陷，本研究旨在建立并验证一种免培养即时型幽门螺杆菌表型药敏新技术。 方法：纳入40例确诊Hp感染的患者，取其胃窦黏膜活检组织，经机械匀浆制备菌悬液；分别加入参照欧洲药敏试验委员会（EUCAST）临界标准配制的阿莫西林、克拉霉素、左氧氟沙星检测体系，采用双荧光探针分别标记死菌与活菌，同时设置无抗生素空白对照组，于37℃孵育 60 min 后使用酶标仪定量检测荧光发射强度，结合相对空白对照组的荧光强度变化判定菌株敏感或耐药。以传统培养 E-test 法为金标准，计算该荧光快速现场评估（Fluorescence rapid on-site evaluation，F-ROSE）法的灵敏度、特异度、Cohen′s kappa 值及与E-test 法的检测一致率，评价其诊断效能。 结果：40例标本中，F-ROSE检测法均检测出Hp阳性，同时完成了对三种抗生素的药敏判定，但其中有14例样本因培养基生长不满意而被剔除，最终每种抗生素各纳入26例样本进行效能评估。F-ROSE法与E-test法的总体符合率：阿莫西林为84.6%（22/26），左氧氟沙星为76.9%（20/26），克拉霉素为69.2%（18/26）。三种药物的Cohen′s kappa系数分别为0.523（0.093–0.953）、0.539（0.215–0.862）和0.412（0.074–0.751），均达到中度一致水平。 结论：本研究首次构建了Hp免培养快速表型药敏（F-ROSE）检测体系，仅需约1小时即可完成菌株药敏评估，突破传统培养法耗时数日的长期技术瓶颈，可实现快速诊断、指导个体化靶向根除方案，有望扭转临床先行经验用药、等待药敏结果滞后的诊疗困境；但本研究样本量有限，后续仍需开展大样本前瞻性研究优化检测参数，并通过随机对照试验验证该技术对 Hp 根除疗效等实际临床结局的改善作用，进一步证实其临床推广价值。

  关键词： 幽门螺杆菌; 抗生素药敏试验; 表型药敏; 培养; 荧光快速现场评估; 快速诊断

   

  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

  提交时间：2026-08-26

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• 序号	提交日期	编号	操作
  1	2026-07-05	10.12201/bmr.202607.00011V1	下载

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