Rödel J, Bohnert J A, Stoll S, Wassill L, Edel B, Karrasch M, Löffler B, Pfister W
Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.
Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany.
Eur J Clin Microbiol Infect Dis. 2017 Jun;36(6):1033-1040. doi: 10.1007/s10096-016-2888-1. Epub 2017 Jan 6.
The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.
使用分子检测方法直接从阳性血培养物（BCs）中快速鉴定病原体和耐药基因，有助于缩短血流感染的诊断时间。在这项研究中，对环介导等温扩增（LAMP）检测方法在血培养诊断中的潜在应用进行了研究。应用了三种不同的检测方法。市售的eazyplex® MRSA检测可检测金黄色葡萄球菌、表皮葡萄球菌、mecA和mecC。开发了两种内部检测方法[革兰氏阳性（GP）和革兰氏阴性（GN）]，用于检测链球菌、肠球菌、vanA、vanB、假单胞菌属、肠杆菌科以及bla家族。共分析了370份阳性血培养物。包括样品制备在内，30分钟内获得了LAMP检测结果。通过实时荧光检测测量扩增情况。荧光强度值的阈值时间为6.25至13.75分钟。检测方法的特异性和敏感性因靶标而异。总体而言，与常规标准诊断相比，87.7%的血培养物获得了真阳性结果。由于缺乏合适的靶标，21次检测为真阴性（5.7%）。耐药基因阳性检测结果与后续抗生素敏感性试验的一致性为100%。在15瓶混合培养的血培养物中，eazyplex®检测在73%的病例中产生了正确结果。这项研究表明，LAMP检测是用于快速血培养检测的快速且节省成本的工具，以便加快诊断报告并改善脓毒症患者的抗生素管理。
