采用血浆宏基因组测序技术检测和定量 ICU 疑似脓毒症患者的细菌 DNA：一项原理验证研究。

Plasma metagenomic sequencing to detect and quantify bacterial DNA in ICU patients suspected of sepsis: A proof-of-principle study.

机构信息

From the Center for Noninvasive Diagnostics (M.T.K., A.O.-B., H.M., S.N.S., T.C.-C., M.M.), Translational Genomics Research Institute, Phoenix; Department of Surgery (M.T.K., B.J., Z.K., T.O., P.R.), University of Arizona, Tucson, Arizona; Department of Surgery (M.T.K., R.A., A.S.), Brigham and Women's Hospital, Boston, Massachusetts; and Pathogen and Microbiome Institute, Northern Arizona University (P.K.), Flagstaff, Arizona.

出版信息

J Trauma Acute Care Surg. 2021 Dec 1;91(6):988-994. doi: 10.1097/TA.0000000000003396.

DOI:10.1097/TA.0000000000003396

PMID:34510074

Abstract

BACKGROUND

Timely recognition of sepsis and identification of pathogens can improve outcomes in critical care patients but microbial cultures have low accuracy and long turnaround times. In this proof-of-principle study, we describe metagenomic sequencing and analysis of nonhuman DNA in plasma. We hypothesized that quantitative analysis of bacterial DNA (bDNA) levels in plasma can enable detection and monitoring of pathogens.

METHODS

We enrolled 30 patients suspected of sepsis in the surgical trauma intensive care unit and collected plasma samples at the time of diagnostic workup for sepsis (baseline), and 7 days and 14 days later. We performed metagenomic sequencing of plasma DNA and used computational classification of sequencing reads to detect and quantify total and pathogen-specific bDNA fraction. To improve assay sensitivity, we developed an enrichment method for bDNA based on size selection for shorter fragment lengths. Differences in bDNA fractions between samples were evaluated using t test and linear mixed-effects model, following log transformation.

RESULTS

We analyzed 72 plasma samples from 30 patients. Twenty-seven samples (37.5%) were collected at the time of infection. Median total bDNA fraction was 1.6 times higher in these samples compared with samples with no infection (0.011% and 0.0068%, respectively, p < 0.001). In 17 patients who had active infection at enrollment and at least one follow-up sample collected, total bDNA fractions were higher at baseline compared with the next sample (p < 0.001). Following enrichment, bDNA fractions increased in paired samples by a mean of 16.9-fold. Of 17 samples collected at the time when bacterial pathogens were identified, we detected pathogen-specific DNA in 13 plasma samples (76.5%).

CONCLUSION

Bacterial DNA levels in plasma are elevated in critically ill patients with active infection. Pathogen-specific DNA is detectable in plasma, particularly after enrichment using selection for shorter fragments. Serial changes in bDNA levels may be informative of treatment response.

LEVEL OF EVIDENCE

Epidemiologic/Prognostic, Level V.

摘要

背景

及时识别脓毒症并鉴定病原体可以改善重症监护患者的预后，但微生物培养的准确性低，且周转时间长。在这项原理验证研究中，我们描述了非人类 DNA 血浆宏基因组测序和分析。我们假设血浆中细菌 DNA（bDNA）水平的定量分析可以实现病原体的检测和监测。

方法

我们纳入了 30 名疑似外科创伤重症监护病房脓毒症的患者，并在脓毒症诊断性检查时（基线）以及 7 天和 14 天后采集血浆样本。我们对血浆 DNA 进行了宏基因组测序，并使用测序reads 的计算分类来检测和定量总 bDNA 分数和病原体特异性 bDNA 分数。为了提高检测灵敏度，我们开发了一种基于较短片段长度的大小选择的 bDNA 富集方法。使用 t 检验和线性混合效应模型评估样本之间 bDNA 分数的差异，并进行对数转换。

结果

我们分析了 30 名患者的 72 个血浆样本。27 个样本（37.5%）在感染时采集。与无感染的样本相比，这些样本的总 bDNA 分数中位数高 1.6 倍（分别为 0.011%和 0.0068%，p<0.001）。在 17 名在入组时存在活动性感染且至少采集了一个后续样本的患者中，与下一个样本相比，基线时总 bDNA 分数更高（p<0.001）。经富集后，配对样本中 bDNA 分数平均增加了 16.9 倍。在 17 个在细菌病原体被鉴定时采集的样本中，我们在 13 个血浆样本中检测到了病原体特异性 DNA（76.5%）。