The Accuracy of Melting Curve–Based Multiplex Real-Time PCR for Diagnosing Helicobacter pylori Resistance to Clarithromycin and Levofloxacin in Stool Specimens

IF 4.3 2区医学 Q1 GASTROENTEROLOGY & HEPATOLOGY

Helicobacter Pub Date : 2025-02-11 DOI:10.1111/hel.70012

Youhua Wang, Xiaoling Gong, Qirui Lai, Hui Fang, Xiyin Yang, Xuan Li, Dong Sheng Liu, Yong Xie

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Abstract

Aims

This study evaluates the accuracy of melting curve–based multiplex real-time PCR (multiplex rt-PCR) on stool samples for diagnosing antibiotic resistance in Helicobacter pylori (H. pylori) compared to E-test and sequencing.

Methods

Gastric biopsies and stool samples were collected from 385 H. pylori-infected patients. A total of 325 strains were isolated, and genomic DNA was extracted from all 385 stool samples. E-tests were conducted to detect phenotypic resistance for clarithromycin and levofloxacin. Sanger sequencing and multiplex rt-PCR were employed to identify H. pylori 23S rRNA and GyrA mutations.

Results

E-test results indicated that 203 (62.5%) were susceptible to both clarithromycin and levofloxacin, 33 (10.2%) exhibited mono-resistance to clarithromycin, 48 (14.8%) showed mono-resistance to levofloxacin, and 41 (12.6%) had dual resistance to both antibiotics. Compared to E-test results, the sensitivity and specificity of the multiplex rt-PCR method for detecting clarithromycin resistance mutation were 93.2 (95% CI 84.3–97.5) and 87.1% (95% CI 82.2–90.9), respectively. For levofloxacin resistance mutation, the multiplex rt-PCR method showed a sensitivity of 80.7 (95% CI 70.3–88.3) and a specificity of 93.0% (95% CI 88.7–95.8). Compared to Sanger sequencing, the sensitivity and specificity of the multiplex rt-PCR method for detecting clarithromycin resistance mutation were 95.8 (95% CI 90.0–98.4) and 96.0% (95% CI 92.6–98.0), respectively. For levofloxacin resistance mutation, the multiplex rt-PCR method showed a sensitivity of 91.3% (95% CI, 83.1–95.9) and a specificity of 96.1% (95% CI, 92.7–98.0).

Conclusion

Genotypic methods, including Sanger sequencing and multiplex rt-PCR, were rapid and reliable for diagnosing clarithromycin and levofloxacin resistance in the stool samples.

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熔融曲线多重实时荧光定量PCR诊断粪便标本中幽门螺杆菌对克拉霉素和左氧氟沙星耐药的准确性

目的与E-test和测序技术相比，评价基于熔融曲线的多重实时荧光定量PCR （multiplex rt-PCR）在粪便样品中诊断幽门螺杆菌（h.p ylori）耐药性的准确性。方法对385例幽门螺旋杆菌感染患者进行胃活检和粪便标本采集。共分离出325株菌株，并从所有385份粪便样本中提取基因组DNA。采用e -试验检测克拉霉素和左氧氟沙星的表型耐药。采用Sanger测序和多重rt-PCR检测幽门螺杆菌23S rRNA和GyrA突变。结果e检验结果显示，对克拉霉素和左氧氟沙星均敏感的203例（62.5%），对克拉霉素单耐药的33例（10.2%），对左氧氟沙星单耐药的48例（14.8%），对两种抗生素双耐药的41例（12.6%）。与e检验结果相比，多重rt-PCR检测克拉霉素耐药突变的敏感性为93.2 (95% CI 84.3 ~ 97.5)，特异性为87.1% （95% CI 82.2 ~ 90.9）。对于左氧氟沙星耐药突变，多重rt-PCR方法的敏感性为80.7 (95% CI 70.3 ~ 88.3)，特异性为93.0% （95% CI 88.7 ~ 95.8）。与Sanger测序相比，多重rt-PCR检测克拉霉素耐药突变的敏感性为95.8 (95% CI 90.0 ~ 98.4)，特异性为96.0% （95% CI 92.6 ~ 98.0）。对于左氧氟沙星耐药突变，多重rt-PCR方法的敏感性为91.3% (95% CI, 83.1 ~ 95.9)，特异性为96.1% （95% CI, 92.7 ~ 98.0）。结论Sanger测序和多重rt-PCR等基因分型方法可快速、可靠地诊断粪便样品中的克拉霉素和左氧氟沙星耐药性。

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来源期刊

Helicobacter 医学-微生物学

CiteScore

8.40

自引率

9.10%

发文量

审稿时长

2 months

期刊介绍： Helicobacter is edited by Professor David Y Graham. The editorial and peer review process is an independent process. Whenever there is a conflict of interest, the editor and editorial board will declare their interests and affiliations. Helicobacter recognises the critical role that has been established for Helicobacter pylori in peptic ulcer, gastric adenocarcinoma, and primary gastric lymphoma. As new helicobacter species are now regularly being discovered, Helicobacter covers the entire range of helicobacter research, increasing communication among the fields of gastroenterology; microbiology; vaccine development; laboratory animal science.