State Key Laboratory of Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
J Clin Microbiol. 2018 Aug 27;56(9). doi: 10.1128/JCM.00019-18. Print 2018 Sep.
Chronic infection with causes peptic ulcers and stomach cancer in a subset of infected individuals. While standard eradication therapy includes multiple antibiotics, treatment failure due to resistance is an increasing clinical problem. Accurate assessment of antimicrobial resistance has been limited by slow growth and sampling of few isolates per subject. We established a method to simultaneously quantify clarithromycin-resistant (mutant) and -susceptible (wild-type) 23S rRNA gene alleles in both stomach and stool samples using droplet digital PCR (ddPCR). In 49 subjects, we assessed the performance of these assays alongside clarithromycin MIC testing of up to 16 isolates per subject and included both cancer (25 subjects) and noncancer (24 subjects) cases. Gastric ddPCR and culture showed agreement with urea breath test (UBT) detection of infection in 94% and 88% of subjects, respectively, while stool ddPCR showed agreement with UBT in 92% of subjects. Based on MIC testing of 43 culture-positive cases, 20 subjects had only susceptible isolates, 14 had a mix of susceptible and resistant isolates, and 9 had only resistant isolates. ddPCR of gastric samples indicated that 21 subjects had only wild-type alleles, 13 had a mixed genotype, and 9 had only mutant alleles. Stool ddPCR detected mutant alleles in four subjects for which mutant alleles were not detected by stomach ddPCR, and no resistant isolates were cultured. Our results indicate that ddPCR detects clarithromycin resistance-associated genotypes, especially in the context of heteroresistance.
幽门螺杆菌慢性感染可导致部分感染者发生消化性溃疡和胃癌。虽然标准根除疗法包含多种抗生素，但由于耐药性，治疗失败已成为日益严重的临床问题。由于生长缓慢且每个受试者的分离株数量有限，因此对抗生素耐药性的准确评估受到限制。我们建立了一种使用液滴数字 PCR（ddPCR）同时定量胃和粪便样本中克拉霉素耐药（突变）和敏感（野生型）23S rRNA 基因等位基因的方法。在 49 名受试者中，我们评估了这些检测方法与每个受试者多达 16 个分离株的克拉霉素 MIC 检测的性能，并包括了癌症（25 名受试者）和非癌症（24 名受试者）病例。胃 ddPCR 和培养与尿素呼气试验（UBT）检测感染的一致性分别为 94%和 88%，而粪便 ddPCR 与 UBT 的一致性为 92%。基于 43 例培养阳性病例的 MIC 检测，20 例仅存在敏感分离株，14 例存在敏感和耐药分离株的混合，9 例仅存在耐药分离株。胃样本的 ddPCR 表明，21 例受试者仅存在野生型等位基因，13 例存在混合基因型，9 例仅存在突变型等位基因。ddPCR 检测到 4 例粪便样本中未检测到突变型等位基因的突变型等位基因，且未培养出耐药分离株。我们的结果表明，ddPCR 可检测克拉霉素耐药相关基因型，尤其是在异质性耐药的情况下。
