Department of Biotechnology, Inland Norway University of Applied Sciences, Holsetgata 22, Hamar, 2317, Norway.
Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
Eur J Clin Microbiol Infect Dis. 2024 Nov;43(11):2177-2190. doi: 10.1007/s10096-024-04929-1. Epub 2024 Sep 16.
Urinary Tract Infections (UTIs) are among the most prevalent infections globally. Every year, approximately 150 million people are diagnosed with UTIs worldwide. The current state-of-the-art diagnostic methods are culture-based and have a turnaround time of 2-4 days for pathogen identification and susceptibility testing.
This study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Urine samples were spiked at clinically significant concentrations of 10-10 CFU/ml. Three DNA extraction kits (BioStic, PowerFood, and Blood and Tissue) were investigated based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. After DNA extraction, the samples were sequenced using MinION and Flongle flow cells.
The Blood and Tissue kit outperformed the other kits based on the investigated parameters. Using nanopore sequencing, all the pathogens and corresponding genes were only identified at a spike concentration of 10 CFU/ml, achieved after 10 min and 3 hours of sequencing, respectively. However, some pathogens and antibiotic-resistance genes (ARG) could be identified from spikes at 10 colony formation units (CFU/mL). The overall turnaround time was five hours, from sample preparation to sequencing-based identification of pathogen ID and antimicrobial resistance genes.
This study demonstrates excellent promise in reducing the time required for informed antibiotic administration from 48 to 72 h to five hours, thereby reducing the number of empirical doses and increasing the chance of saving lives.
尿路感染（UTI）是全球最常见的感染之一。每年，全球约有 1.5 亿人被诊断患有 UTI。目前最先进的诊断方法基于培养，需要 2-4 天才能确定病原体并进行药敏试验。
本研究首先建立了一种基于光学密度的方法，用六种最常见的尿路病原体对健康尿液样本进行接种。尿液样本以 10-10 CFU/ml 的临床显著浓度接种。根据 DNA 产量、平均处理时间、洗脱体积和每次提取的平均成本，研究了三种 DNA 提取试剂盒（BioStic、PowerFood 和 Blood and Tissue）。DNA 提取后，使用 MinION 和 Flongle 流动池对样本进行测序。
Blood and Tissue 试剂盒在研究的参数方面优于其他试剂盒。使用纳米孔测序，所有病原体和相应基因仅在 10 CFU/ml 的接种浓度下被识别，分别需要 10 分钟和 3 小时的测序时间。然而，一些病原体和抗生素耐药基因（ARG）可以从 10 个菌落形成单位（CFU/ml）的接种物中识别出来。从样品制备到基于测序的病原体 ID 和抗菌药物耐药基因鉴定，总周转时间为五个小时。
本研究表明，在减少从 48 小时到 72 小时的知情抗生素给药所需时间方面具有巨大的潜力，从而减少了经验剂量的数量，增加了挽救生命的机会。
