Table_1_Comprehensive evaluation of plasma microbial cell-free DNA sequencing for predicting bloodstream and local infections in clinical practice: a multicenter retrospective study.docx

BackgroundMetagenomic next-generation sequencing (mNGS) of plasma cell-free DNA (cfDNA) shows promising application for complicated infections that cannot be resolved by conventional microbiological tests (CMTs). The criteria for cfDNA sequencing are currently in need of agreement and standardization.
MethodsWe performed a retrospective cohort observation of 653 patients who underwent plasma cfDNA mNGS, including 431 with suspected bloodstream infections (BSI) and 222 with other suspected systemic infections. Plasma mNGS and CMTs were performed simultaneously in clinical practice. The diagnostic efficacy of plasma mNGS and CMTs in the diagnosis of blood-borne and other systemic infections was evaluated using receiver operating characteristic (ROC) curves. The sensitivity and specificity of the two methods were analyzed based on the final clinical outcome as the gold standard.
ResultsThe mNGS test showed an overall positive rate of 72.3% (472/653) for detecting microorganisms in plasma cfDNA, with a range of 2 to 6 different microorganisms detected in 171 patient specimens. Patients with positive mNGS results were more immunocompromised and had a higher incidence of severe disease (P<0·05). The sensitivity of mNGS was higher for BSI (93·5%) and other systemic infections (83·6%) compared to CMTs (37·7% and 14·3%, respectively). The mNGS detected DNA from a total of 735 microorganisms, with the number of microbial DNA reads ranging from 3 to 57,969, and a higher number of reads being associated with clinical infections (P<0·05). Of the 472 patients with positive mNGS results, clinical management was positively affected in 203 (43%) cases. Negative mNGS results led to a modified clinical management regimen in 92 patients (14.1%). The study also developed a bacterial and fungal library for plasma mNGS and obtained comparisons of turnaround times and detailed processing procedures for rare pathogens.
ConclusionOur study evaluates the clinical use and analytic approaches of mNGS in predicting bloodstream and local infections in clinical practice. Our results suggest that mNGS has higher positive predictive values (PPVs) for BSI and systemic infections compared to CMTs, and can positively affect clinical management in a significant number of patients. The standardized whole-process management procedure for plasma mNGS developed in this study will ensure improved pre-screening probabilities and yield clinically valuable data.

背景 血浆游离DNA（cell-free DNA, cfDNA）的宏基因组下一代测序（metagenomic next-generation sequencing, mNGS）在传统微生物检测（conventional microbiological tests, CMTs）无法明确诊断的复杂感染中展现出良好的应用前景。当前，cfDNA测序的相关标准仍缺乏统一共识与标准化规范。
方法 本研究对653例行血浆cfDNA mNGS检测的患者开展回顾性队列观察研究，其中431例疑似血流感染（bloodstream infections, BSI），222例疑似其他全身性感染。临床实践中，血浆mNGS与CMTs同步开展检测。以最终临床结局为金标准，采用受试者工作特征（receiver operating characteristic, ROC）曲线评估血浆mNGS与CMTs在血源感染及其他全身性感染诊断中的诊断效能，并基于金标准分析两种方法的灵敏度与特异度。
结果 血浆cfDNA mNGS检测的微生物总阳性率为72.3%（472/653），其中171份患者标本可检出2~6种不同微生物。mNGS结果阳性的患者多存在免疫功能低下情况，且重症疾病发生率更高（P<0.05）。相较于CMTs（血流感染组37.7%、其他全身性感染组14.3%），mNGS对血流感染（93.5%）及其他全身性感染（83.6%）的灵敏度更高。mNGS共检出735种微生物的DNA，其序列读段数范围为3~57969，且读段数越高与临床感染的相关性越强（P<0.05）。在472例mNGS结果阳性的患者中，203例（43%）的临床诊疗方案得到积极优化。92例（14.1%）mNGS结果阴性的患者调整了临床诊疗方案。本研究还构建了血浆mNGS专用细菌与真菌数据库，并对比了检测周转时间，同时梳理了罕见病原体的详细处理流程。
结论 本研究评估了血浆mNGS在临床实践中诊断血流感染与局部感染的临床应用价值及分析方法。研究结果显示，相较于CMTs，mNGS对血流感染及全身性感染具有更高的阳性预测值（positive predictive values, PPVs），且可显著改善大量患者的临床诊疗结局。本研究构建的标准化血浆mNGS全流程管理规范，可提升前置筛查效能并获得具有临床价值的检测数据。