Zelenin Sergey, Ramachandraiah Harisha, Faridi Asim, Russom Aman
Science for Life Laboratory, Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, PO Box 1031, 17121, Solna, Stockholm, Sweden.
Methods Mol Biol. 2017;1547:175-186. doi: 10.1007/978-1-4939-6734-6_14.
Bacterial blood stream infection (BSI) potentially leads to life-threatening clinical conditions and medical emergencies such as severe sepsis, septic shock, and multi organ failure syndrome. Blood culturing is currently the gold standard for the identification of microorganisms and, although it has been automated over the decade, the process still requires 24-72 h to complete. This long turnaround time, especially for the identification of antimicrobial resistance, is driving the development of rapid molecular diagnostic methods. Rapid detection of microbial pathogens in blood related to bloodstream infections will allow the clinician to decide on or adjust the antimicrobial therapy potentially reducing the morbidity, mortality, and economic burden associated with BSI. For molecular-based methods, there is a lot to gain from an improved and straightforward method for isolation of bacteria from whole blood for downstream processing.We describe a microfluidic-based sample-preparation approach that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.
细菌血流感染（BSI）可能导致危及生命的临床状况和医疗紧急情况，如严重脓毒症、感染性休克和多器官功能衰竭综合征。血培养是目前鉴定微生物的金标准，尽管在过去十年中已实现自动化，但该过程仍需24至72小时才能完成。这种较长的周转时间，尤其是对于抗菌药物耐药性的鉴定，推动了快速分子诊断方法的发展。快速检测与血流感染相关的血液中的微生物病原体，将使临床医生能够决定或调整抗菌治疗，有可能降低与BSI相关的发病率、死亡率和经济负担。对于基于分子的方法，改进并简化从全血中分离细菌以进行下游处理的方法将大有裨益。我们描述了一种基于微流控的样品制备方法，该方法能快速、选择性地裂解所有血细胞，同时提取完整细菌用于下游分析。全血先接触一种温和的去污剂，裂解大多数血细胞，然后用去离子水进行渗透压休克，以消除剩余的白细胞。回收的细菌具有100%的活力，这为进行药敏试验和基于核酸的分子鉴定开辟了可能性。
