Dalla-Costa Libera M, Morello Luis G, Conte Danieli, Pereira Luciane A, Palmeiro Jussara K, Ambrosio Altair, Cardozo Dayane, Krieger Marco A, Raboni Sonia M
Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil; Laboratory of Bacteriology, Universidade Federal do Paraná, Rua Padre Camargo, 280, - 80060-240, Curitiba, Brazil; Faculdades e Instituto de Pesquisa Pelé Pequeno Príncipe, Av. Silva Jardim, 1632, - 80250-200, Curitiba, Brazil.
Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil; Laboratory of Functional Genomics, Instituto Carlos Chagas, Fundação Oswaldo Cruz, Rua Professor Algacyr Munhoz Mader, 3775, - 81310-020, Curitiba, Brazil.
J Microbiol Methods. 2017 Sep;140:61-66. doi: 10.1016/j.mimet.2017.06.020. Epub 2017 Jun 29.
Sepsis is the leading cause of death in intensive care units (ICUs) worldwide and its diagnosis remains a challenge. Blood culturing is the gold standard technique for blood stream infection (BSI) identification. Molecular tests to detect pathogens in whole blood enable early use of antimicrobials and affect clinical outcomes. Here, using real-time PCR, we evaluated DNA extraction using seven manual and three automated commercially available systems with whole blood samples artificially contaminated with Escherichia coli, Staphylococcus aureus, and Candida albicans, microorganisms commonly associated with BSI. Overall, the commercial kits evaluated presented several technical limitations including long turnaround time and low DNA yield and purity. The performance of the kits was comparable for detection of high microorganism loads (10CFU/mL). However, the detection of lower concentrations was variable, despite the addition of pre-processing treatment to kits without such steps. Of the evaluated kits, the UMD-Universal CE IVD kit generated a higher quantity of DNA with greater nucleic acid purity and afforded the detection of the lowest microbial load in the samples. The inclusion of pre-processing steps with the kit seems to be critical for the detection of microorganism DNA directly from whole blood. In conclusion, future application of molecular techniques will require overcoming major challenges such as the detection of low levels of microorganism nucleic acids amidst the large quantity of human DNA present in samples or differences in the cellular structures of etiological agents that can also prevent high-quality DNA yields.
脓毒症是全球重症监护病房（ICU）中主要的死亡原因，其诊断仍然是一项挑战。血培养是血流感染（BSI）鉴定的金标准技术。检测全血中病原体的分子检测方法能够早期使用抗菌药物并影响临床结果。在此，我们使用实时聚合酶链反应（PCR），评估了七种手动和三种自动市售系统对人工污染了大肠杆菌、金黄色葡萄球菌和白色念珠菌（这些都是与BSI常见相关的微生物）的全血样本进行DNA提取的情况。总体而言，所评估的商业试剂盒存在一些技术局限性，包括周转时间长以及DNA产量和纯度低。这些试剂盒在检测高微生物载量（10CFU/mL）时性能相当。然而，尽管对没有此类步骤的试剂盒添加了预处理，但较低浓度的检测结果却各不相同。在所评估的试剂盒中，UMD通用CE体外诊断试剂盒产生的DNA量更高，核酸纯度更高，并且能够检测样本中最低的微生物载量。该试剂盒包含预处理步骤似乎对于直接从全血中检测微生物DNA至关重要。总之，分子技术的未来应用将需要克服重大挑战，例如在样本中大量人类DNA存在的情况下检测低水平的微生物核酸，或者病原体细胞结构的差异也可能阻碍高质量DNA产量。
