Binmaeil Hasyanee, Hanafiah Alfizah, Mohamed Rose Isa, Raja Ali Raja Affendi
Department of Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia.
GUT Research Group, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia.
Infect Drug Resist. 2021 Oct 6;14:4129-4145. doi: 10.2147/IDR.S325056. eCollection 2021.
More than half of the world's population is infected with , which can cause chronic gastritis. WHO has regarded clarithromycin-resistant as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin- and levofloxacin-resistant strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in directly from the gastric biopsies.
Specific primers and probes were designed to amplify and mutations in 23S rRNA and genes. Singleplex and triplex qPCR assays were optimized and the assay's sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies.
In this study, 14.7% (84/571) of the gastric biopsies were positive for by conventional methods and 23.8% (136/571) were positive by the -qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and -LR were 8.72 and 0.04, respectively. The -positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of , respectively.
The effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin- and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of infection at a much greater pace.
世界上超过一半的人口感染了幽门螺杆菌，它可导致慢性胃炎。世界卫生组织已将耐克拉霉素的幽门螺杆菌视为高度优先关注的病原体。因此，准确诊断和检测耐克拉霉素和左氧氟沙星的幽门螺杆菌菌株对于感染的恰当管理至关重要。本研究的目的是开发并优化多重定量PCR检测方法，以直接从胃活检组织中检测与克拉霉素和左氧氟沙星耐药相关的突变。
设计特异性引物和探针，用于扩增23S rRNA和gyrA基因中的幽门螺杆菌及突变。对单重和三重qPCR检测方法进行优化，并确定检测方法的敏感性和特异性。在571份胃活检组织上进行优化后的多重qPCR检测。
在本研究中，通过传统方法检测，14.7%（84/571）的胃活检组织幽门螺杆菌呈阳性，通过幽门螺杆菌qPCR检测为23.8%（136/571）呈阳性，敏感性为96.4%，特异性为88.5%，阳性似然比和阴性似然比分别为8.72和0.04。对幽门螺杆菌阳性样本（n = 136）进行多重qPCR检测，检测到23S rRNA基因中的A2142G和A2143G突变（20.6%，28/136）导致克拉霉素耐药，以及gyrA基因突变N87K、N87I、D91N和D91Y（11.8%，16/136）导致左氧氟沙星耐药。23S rRNA基因qPCR的敏感性和特异性分别为100%和98.7%，而gyrA基因qPCR的敏感性和特异性分别为100%和99.8%。
该qPCR检测方法的有效性在于它在检测低细菌载量时具有敏感性，有助于及时检测耐克拉霉素和左氧氟沙星的菌株，尤其是在混合感染的情况下。由于它不依赖培养，可为临床医生提供一线治疗中应使用的抗生素信息，从而以更快的速度改善幽门螺杆菌感染的管理。
