Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan.
J Med Microbiol. 2019 Feb;68(2):169-177. doi: 10.1099/jmm.0.000903. Epub 2019 Jan 9.
In infectious disease therapy, administration of adequate antimicrobial agents is essential for preventing the emergence and spread of resistant bacteria. However, conventional antimicrobial susceptibility testing (AST), based on bacterial growth, is time consuming; therefore, a rapid, simple assay is needed for the timely selection of appropriate antibiotics in clinical laboratories. Here, we established a simple, cost-effective, time-saving and highly sensitive AST assay based on loop-mediated isothermal amplification (LAMP).
The targeted bacteria were cultivated for a short period with or without antibiotic before the LAMP reaction. The time to detect a positive reaction with LAMP was used to generate a threshold time (Tt) value, and subtraction of the Tt value for an antibiotic-free sample from the Tt value in an antibiotic-exposed sample generated the ΔTt value, which was used as a marker of antimicrobial susceptibility. The ΔTt value generated using the LAMP-based assay simply and quickly detected antimicrobial resistance in clinical Escherichia coli isolates.
Detection of susceptibility to levofloxacin using the ΔTt value perfectly matched with the results of the conventional assay. In addition, the sensitivity and specificity for the detection of ampicillin, trimethoprim-sulfamethoxazole and fosfomycin resistance were 100 %, 93.8 %, 100 % and 80.0 %, 93.3 %, 97.6 %, respectively.
These results showed that this LAMP-based AST has high sensitivity and specificity for detecting resistant strains and a significant time advantage compared with the conventional method.
在传染病治疗中，给予足够的抗菌药物对于防止耐药菌的出现和传播至关重要。然而，基于细菌生长的传统抗菌药物敏感性测试（AST）耗时较长；因此，临床实验室需要一种快速、简单的方法来及时选择合适的抗生素。在这里，我们建立了一种基于环介导等温扩增（LAMP）的简单、经济、省时且高度敏感的 AST 检测方法。
在进行 LAMP 反应之前，将目标细菌短时间培养并加入或不加入抗生素。使用 LAMP 检测阳性反应的时间来生成阈值时间（Tt）值，并用抗生素处理样本的 Tt 值减去无抗生素样本的 Tt 值生成 ΔTt 值，该值可作为抗菌药物敏感性的标志物。基于 LAMP 的检测方法产生的 ΔTt 值可简单快速地检测临床大肠杆菌分离株的抗菌药物耐药性。
使用 ΔTt 值检测左氧氟沙星的敏感性与传统检测方法的结果完全一致。此外，检测氨苄西林、甲氧苄啶-磺胺甲恶唑和磷霉素耐药性的敏感性和特异性分别为 100%、93.8%、100%和 80.0%、93.3%、97.6%。
这些结果表明，与传统方法相比，这种基于 LAMP 的 AST 具有高灵敏度和特异性，可用于检测耐药菌株，并具有显著的时间优势。
