Sanabria Adriana, Hjerde Erik, Johannessen Mona, Sollid Johanna Ericson, Simonsen Gunnar Skov, Hanssen Anne-Merethe
Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, UiT - The Arctic University of Norway, Tromsø, Norway.
Department of Chemistry, Centre for Bioinformatics, UiT - The Arctic University of Norway, Tromsø, Norway.
Front Microbiol. 2020 Jul 17;11:1687. doi: 10.3389/fmicb.2020.01687. eCollection 2020.
Clinical metagenomics is actively moving from research to clinical laboratories. It has the potential to change the microbial diagnosis of infectious diseases, especially when detection and identification of pathogens can be challenging, such as in prosthetic joint infection (PJI). The application of metagenomic sequencing to periprosthetic joint tissue (PJT) specimens is often challenged by low bacterial load in addition to high level of inhibitor and contaminant host DNA, limiting pathogen recovery. Shotgun-metagenomics (SMg) performed directly on positive blood culture bottles (BCBs) inoculated with PJT may be a convenient approach to overcome these obstacles. The aim was to test if it is possible to perform SMg on PJT inoculated into BCBs for pathogen identification in PJI diagnosis. Our study was conducted as a laboratory method development. For this purpose, spiked samples (positive controls), negative control and clinical tissue samples (positive BCBs) were included to get a comprehensive overview. We developed a method for preparation of bacterial DNA directly from PJT inoculated in BCBs. Samples were processed using MolYsis5 kit for removal of human DNA and DNA extracted with BiOstic kit. High DNA quantity/quality was obtained, and no inhibition was observed during the library preparation, allowing further sequencing process. DNA sequencing reads obtained from the BCBs, presented a low proportion of human reads (<1%) improving the sensitivity of bacterial detection. We detected a 19-fold increase in the number of reads mapping to human in a sample untreated with MolYsis5. Taxonomic classification of clinical samples identified a median of 96.08% (IQR, 93.85-97.07%; range 85.7-98.6%) bacterial reads. Shotgun-metagenomics results were consistent with the results from a conventional BCB culture method, validating our approach. Overall, we demonstrated a proof of concept that it is possible to perform SMg directly on BCBs inoculated with PJT, with potential of pathogen identification in PJI diagnosis. We consider this a first step in research efforts needed to face the challenges presented in PJI diagnoses.
临床宏基因组学正积极地从研究领域转向临床实验室。它有潜力改变传染病的微生物诊断，尤其是在病原体的检测和鉴定具有挑战性时，例如在人工关节感染（PJI）中。除了高水平的抑制剂和污染性宿主DNA外，宏基因组测序在假体周围关节组织（PJT）标本中的应用还常常受到细菌载量低的挑战，限制了病原体的回收。直接对接种了PJT的阳性血培养瓶（BCB）进行鸟枪法宏基因组学（SMg）可能是克服这些障碍的便捷方法。目的是测试对接种到BCB中的PJT进行SMg以鉴定PJI诊断中的病原体是否可行。我们的研究作为一种实验室方法开发进行。为此，纳入了加标样本（阳性对照）、阴性对照和临床组织样本（阳性BCB）以获得全面的概述。我们开发了一种直接从接种在BCB中的PJT制备细菌DNA的方法。使用MolYsis5试剂盒处理样本以去除人类DNA，并用BiOstic试剂盒提取DNA。获得了高数量/质量的DNA，并且在文库制备过程中未观察到抑制作用，从而允许进一步的测序过程。从BCB获得的DNA测序读数显示人类读数的比例较低（<1%），提高了细菌检测的灵敏度。我们发现在未用MolYsis5处理的样本中，映射到人类的读数数量增加了19倍。临床样本的分类学分类确定细菌读数的中位数为96.08%（四分位间距，93.85 - 97.07%；范围85.7 - 98.6%）。鸟枪法宏基因组学结果与传统BCB培养方法的结果一致，验证了我们的方法。总体而言，我们证明了一个概念验证，即可以直接对接种了PJT的BCB进行SMg，具有在PJI诊断中鉴定病原体的潜力。我们认为这是应对PJI诊断中提出的挑战所需研究工作的第一步。
