宏基因二代测序技术对非结核分枝杆菌感染病原学诊断的价值

目的:探讨宏基因二代测序(metagenomic next-generation sequencing,mNGS)技术对非结核分枝杆菌(non-tuberculosis mycobacteria,NTM)感染病原学诊断的价值。方法:回顾性选取2018年9月至2019年12月复旦大学附属中山医院感染病科收治的疑似NTM感染患者183例,共计样本388例,比较常规微生物培养法和mNGS技术对NTM病原体的检出率差异及不同样本类型的亚组分析。结果:常规微生物培养法和mNGS总体检测阳性率差异无统计学意义(21.9%vs 24.7%)。痰液样本培养法的阳性率明显高于mNGS(56.6%vs 22.9%,P0.000 1),而组织样本却低于mNGS(7.8%vs 27.8%,P0.000 1)。另外,mNGS对患者抗菌药物调整频率低于培养法(20.3%vs 40.2%,P0.05)。结论:mNGS对NTM灵敏度较低,需重点改进以促进此技术更好地应用于临床。     

宏基因二代测序技术对肝脓肿病原学诊断的价值

目的:探讨宏基因二代测序(metagenomics next-generation sequencing,mNGS)技术对肝脓肿病原学诊断的价值。方法:前瞻性纳入2020年2月至2021年4月复旦大学附属中山医院急诊科收治的35例肝脓肿患者,采用常规微生物培养法和mNGS技术分别检测血液和脓肿引流液样本。并将患者按是否出现休克分为两组,采用SPSS 25.0进行统计学分析,对比分析两组患者之间的差异。结果:mNGS技术在血液样本和引流液样本中总体检测阳性率显著高于常规培养法(血液：67.6% vs. 15.2%, P<0.05;引流液：100% vs. 55.2%, P<0.05)。35例肝脓肿病例中71.4%检出致病病原体为肺炎克雷伯菌。休克组患者脓肿引流液样本中mNGS检测出的致病病原体序列数显著高于非休克组( P<0.05)。 结论:mNGS能够快速准确的检测出肝脓肿的致病病原体,为临床精准治疗提供重要的病原学依据。     

宏基因二代测序技术对慢性肺曲霉病病原学诊断的价值

目的:探讨宏基因二代测序(metagenomic next-generation sequencing, mNGS)技术对慢性肺曲霉病(chronic pulmonary aspergillosis, CPA)病原学诊断的价值。方法:回顾性收集2017年9月至2019年9月复旦大学附属中山医院感染病科收治的78例疑似CPA患者病例资料,采集样本进行常规微生物学培养、血清烟曲霉特异性抗体IgG检测和mNGS,比较3种检测方法的灵敏度和特异度。根据诊断标准,将78例疑似CPA患者最终诊断结果分为CPA组(35例)和非CPA组(43例)。结果:以最终诊断结果为金标准,常规培养法、烟曲霉特异性抗体IgG检测和mNGS的灵敏度分别为28.6%、48.6%和65.7%,特异度分别为93.0%、90.7%和86.0%,其中mNGS的灵敏度显著高于常规培养法(P=0.001)。ROC曲线下面积分别为0.759、0.696和0.608。结论:在CPA的诊断中,mNGS的灵敏度显著高于常规培养法和烟曲霉特异性抗体IgG检测,且在检测时间上具有明显优势。     

宏基因组学第二代测序技术对比传统实验室微生物培养在脓毒症病原学诊断中的优势

目的通过对宏基因组学第二代测序技术(mNGS)获得的病原体与实验室培养结果进行对比,了解mNGS在脓毒症病原学诊断中的优势及其临床指导意义。方法将入选的脓毒症患者的标本(肺泡灌洗液、痰液、血液、脑脊液、胸水、腹水、分泌物等)同时送检mNGS和实验室细菌培养,对结果进行对比分析,评价mNGS在脓毒症病原学诊断方面的临床价值。结果mNGS的阳性率为78.9%;细菌培养的阳性率为40.4%(P<0.05)。通过mNGS共检出致病病原体57种,其中细菌31种,真菌16种,病毒7种,非典型病原体3种;细菌培养共检出病原体24种,其中细菌18种,真菌6种。以培养结果为金标准,mNGS的敏感度为76.2%,特异度为29.8%,阳性预测值为42.3%,阴性预测值为64.8%。根据病原学结果的抗生素调整将患者分为三组:按mNGS调整为mNGS组、经验性调整为经验组、按培养结果调整为传统培养组。mNGS组ICU住院时间更短(P<0.05),培养组降钙素原下降更明显(P<0.05)。结论mNGS在感染性疾病病原体的诊断方面较传统微生物培养时间更短,阳性率更高,在少见病原体、罕见病原体诊断方面有显著优势,可缩短患者ICU住院时间。     

宏基因组测序技术在虫媒传播疾病诊断中的应用与思考

虫媒传播疾病是危及生命的严重传染性疾病, 快速且精准病原学诊断是临床及时有效治疗并降低病死率、减少后遗症的前提。虫媒病实验室诊断以靶向性的血清学检测和聚合酶链反应为主, 对少见虫媒病原体检出困难。目前, 宏基因组二代测序技术(mNGS)已从科研走向临床应用。mNGS检测感染标本中全部生物的核酸序列并进行生物信息学分析, 在少见病原体诊断和溯源方面具有显著优势。但同时mNGS也面临着整个操作流程中可能引入的背景微生物污染、数据库限制造成的错误结果、临床治疗亟需的病原体耐药性和宿主免疫状态等信息的挑战。该文主要就mNGS技术、其在虫媒病原体诊断应用及其所面临的挑战与难题等方面进行系统论述。随着mNGS在检测流程、检测结果分析和解读等方面不断优化, 将为临床感染病病原学诊断带来新的发展与革新。     

宏基因组二代测序技术在临床病原学诊断中的应用

感染性疾病是威胁人类生命健康的主要疾病,病原学诊断是感染性疾病诊治的关键环节。近年来,宏基因组二代测序技术(mNGS)迅速发展。与传统病原学检测技术相比,mNGS能无偏倚性直接分析出标本中的病原体谱、丰度和分布情况,改变传统病原学诊断模式,具有良好的应用前景。该文对mNGS在病原体检测、感染性疾病诊断、检测影响因素和结果判读等方面进行评述,并对存在问题和未来发展进行总结,以期促进mNGS技术合理应用。     

脑脊液二代测序在中枢神经系统感染中的病原学诊断价值北大核心CSCD

目的探讨宏基因组二代测序技术(metagenomic next-generation sequencing,mNGS)在中枢神经系统(central nervous system,CNS)感染性疾病中的病原学诊断价值。方法以2018年1月至2020年6月郑州大学第一附属医院收治的中枢神经系统感染患者为研究对象,根据纳入和排除标准最终确定170例患者。收集患者的一般临床资料以及病原体检测结果。纳入患者均送检常规以及mNGS检测,将检测结果分为常规方法检测组及mNGS检测组。符合正态分布的计量资料用均数±标准差(x±s)表示;不符合正态分布的计量资料用中位数及四分位数表示;分类资料采用例数及百分比[n(%)]表示;比较采用χ2检验或Fisher's精确检验;一致性检验以Kappa值表示;对比分析两种方法对病原微生物的检出情况及检出病原谱的规律。结果在CNS感染性疾病中mNGS检测的总体阳性率高于常规方法(58.23%vs.18.82%),且差异有统计学意义(P<0.01)。两种检测方法均阳性的20例样本中,所检出病原体种类完全一致者10例,完全不一致者和部分一致者各5例。在结核性神经系统感染病例检测中,检测阳性率从高到低依次为血T-SPOT、脑脊液mNGS、结核杆菌DNA、腺苷脱氨酶(ADA)以及结核特异性抗原抗体检测,分别为66.7%、53.8%、40.0%、24.0%、4.0%,抗酸染色阳性率为0%;mNGS与常规方法结合的检测阳性率为80.8%。结论脑脊液mNGS在CNS感染中病原菌检出率总体优于常规方法;但对结核杆菌的检测阳性率较T-SPOT未显示出明显优势;mNGS检测的病原菌更为广泛,有助于深入全面地了解中枢神经系统感染的菌种特点,两者结合在一定程度上弥补了临床常规检测的不足,可最大化提升检出率。     

宏基因组二代测序在食管气管瘘气道支架置入术后肺炎病原体检测中的价值

目的 观察食管气管瘘气道支架置入术后肺炎患者支气管肺泡灌洗液(BALF)、防污染样本毛刷(PSB)采样分别行宏基因组二代测序(mNGS)和病原体培养的病原体检出情况，探讨mNGS在病原学诊断中的价值。方法 2020年8月—2021年8月应急总医院诊治食管气管瘘气道支架置入术后肺炎患者48例，抗感染治疗前行支气管镜检查，采集BALF和PSB采样，分别行mNGS及病原体培养检查；采集痰标本行病原体培养检查。比较5种方法病原体检出率；分析5种方法检出病原体种类及分布情况；比较BALF mNGS与PSB采样mNGS检出主要病原体序列数。结果 BALF mNGS病原体检出率(95.83%)与PSB采样mNGS(95.83%)比较差异无统计学意义(χ²<0.001,P>0.999);BALF培养(37.50%)、PSB采样培养(33.33%)、痰培养(33.33%)病原体检出率比较差异无统计学意义(χ²=0.170,P>0.999),均低于BALF mNGS、PSB采样mNGS(P<0.05)。BALF mNGS共检出51种病原体...     

血浆宏基因组二代测序鉴定重型再生障碍性贫血血流感染病原体研究

目的评估无细胞血浆宏基因组二代测序(mNGS)病原体识别对重型再生障碍性贫血(AA)血流感染的诊断意义。方法应用mNGS与常规检测方法(血培养等)同步检测2021年2月至2022年2月中国医学科学院血液病医院贫血诊疗中心连续收治的29例AA患者共33例次送检样本, 评估mNGS与常规检测的诊断一致性、对临床治疗获益的影响及临床准确度。结果①33例次患者经mNGS和常规检测方法检测, 其中25例次(75.76%)检出潜在病原微生物;共检出病原微生物72株, 其中65株(90.28%)仅经mNGS检出。②诊断一致性评估:2例次(6.06%)组合符合(Composite), 18例次(54.55%)mNGS唯一符合(mNGS only), 2例次(6.06%)常规检测方法唯一符合(Conventional testing only), 1例次(3.03%)共同符合(mNGS/Conventional testing), 10例次(30.3%)完全不符合(None)。③临床治疗获益评估:8例次(24.24%)为启动靶向治疗(Initation of targeted treatment), 1...     

宏基因二代测序在脊柱感染诊治中的应用进展

脊柱感染（spinal infection,SI）是一种比较少见的感染性疾病,由于经验性使用抗生素使耐药菌感染的几率增加,同时检测技术的进步导致检出率增加,其发病率呈上升趋势。判断病原体的类型从而针对性地使用抗生素是治疗的关键,然而传统检测方法检出率低且耗时长,不利于脊柱感染的快速精确诊断。宏基因二代测序（metagenomics next-generation sequencing,mNGS）是一种可对样本中所有核酸片段进行测序的检测技术,其出现颠覆了传统检测方法,在脊柱感染的诊断与治疗中发挥重要作用。本文就mNGS在脊柱感染诊治中的应用进展进行综述。     

Aggressive disseminated Rhizomucor pusillus infection in a Ph-like acute lymphoblastic leukemia patient: Early detection by cell-free DNA next-generation sequencing

Disseminated Rhizomucor pusillus infection is a very rare but fatal complication in immunocompromised patients, because of aggressive clinical process with delayed diagnosis by routine laboratory tests. Recently, cell-free DNA next-generation sequencing (cfDNA NGS) has been used for the timely detection of infectious pathogens including mucormycosis. Herein, we described an 18-year-old male with Philadelphia-like acute lymphoblastic leukemia who received a timely diagnosis of R. pusillus infection by cell-free DNA next-generation sequencing, and confirmed by silver staining and qPCR on biopsy tissue. To the best of our knowledge, this was the first case of disseminated R. pusillus infection detected by cfDNA NGS and confirmed by histology in an adult leukemia patient. In addition, this case was supposed to be the most extensive R. pusillus infection diagnosed, involving the lung, skin, liver, kidney, spleen and brain, and the only one case who survived the infection had a favorable outcome through treatment with liposome amphotericin B sequential posaconazole. This case suggested that cfDNA NGS could be used to successfully detect rare pathogen infections, and this was especially important for R. pusillus because timely diagnosis and effective treatment could improve the prognosis of this kind of patient.     

A case of tick-transmitted Q fever in Lishui,China diagnosed by next-generation sequencing

Q fever is a zoonotic disease caused by Coxiella burnetii. Most patients have non-specific symptoms at onset. In addition, routine diagnostic tests for C. burnetii are not sensitive, and the bacterium cannot grow in general culture medium. The diagnosis of Q fever therefore poses a challenge. This case study describes a man with a clear history of tick bite who had recurrent fever, pneumonia, and liver damage. Routine tests and bacterial cultures failed to clarify the pathogeny, but laboratory and imaging data suggested infection. After routine tests were exhausted, we detected the presence of C. burnetii in a whole blood sample using next-generation sequencing (NGS). To our knowledge, this is the first report of Q fever associated with Coxiella burnetii detected directly from blood samples in Lishui, China. NGS has revolutionized the diagnosis of infectious diseases, especially those caused by rare or newly discovered pathogens, and patient responses have finally proved its substantial benefits. NGS has important clinical significance for the early diagnosis of chronic Q fever. This proof-of-concept study is worthy of promotion in clinical practice.     

高通量测序技术在尿路感染及腹膜透析相关腹膜炎病原学诊断中的价值

目的比较尿路感染、腹膜透析相关腹膜炎患者采用高通量测序技术、常规培养法对病原体的检出情况,探讨高通量测序技术在尿路感染、腹膜透析相关腹膜炎病原学诊断中的应用价值。方法收集77例尿路感染患者中段尿标本,36例腹膜透析相关腹膜炎患者透析流出液标本,分别应用高通量测序技术、常规培养法进行检测,记录病原体检出率及病原体分布情况,以培养法为金标准,计算高通量测序法对病原体检测的灵敏度。结果77例尿路感染患者中,高通量测序法检出病原体70例(90.9%),共分离出病原体169株;培养法检出病原体27例(35.1%),共分离出病原体27株;以培养法为金标准,高通量测序法对尿路感染患者病原体检出的灵敏度为96.3%。36例腹膜透析相关腹膜炎患者中,高通量测序法检出病原体28例(77.8%),共分离出病原体39株,培养法检出病原体11例(30.6%),共分离出病原体11株;以培养法为金标准,高通量测序法对腹膜透析相关腹膜炎患者病原体检出的灵敏度为72.7%。结论对尿路感染、腹膜透析相关腹膜炎患者,采用高通量测序技术检出的病原体株数多于培养法,可作为培养法的有效补充。     

Effects of viral infection and microbial diversity on patients with sepsis: A retrospective study based on metagenomic next-generation sequencing

BACKGROUND: The study aims to investigate the performance of a metagenomic next-generation sequencing (NGS)-based diagnostic technique for the identification of potential bacterial and viral infections and effects of concomitant viral infection on the survival rate of intensive care unit (ICU) sepsis patients.     

Evaluation of infections in orthopedic patients using next-generation sequencing

IntroductionCulture tests are used to diagnose infections, but there are various problems such as low sensitivity in detecting infections in orthopedic cases. To address this problem, next generation sequencing (NGS) analysis, which can comprehensively search for bacterial genes, is being applied clinically. In this study, we examined whether NGS analysis was useful in evaluating infections in orthopedic cases.MethodsThe participants were 23 patients suspected of having an infection between 2016 and 2017. Samples were collected from tissues suspected of being infected and were subjected to culture tests and NGS analysis, and the positive rates from the culture tests and from the NGS analysis were compared. We also attempted to determine cutoff value for the NGS analysis.ResultsA total of 20 cases were ultimately diagnosed as infections and 3 cases were diagnosed as non-infections. The sensitivity of the culture tests was 70%, and the sensitivity of the NGS analysis was 55%. When the NGS analysis was performed with the diversity index set to the cut-off value, the sensitivity was 75% for the Simpson index. In this study, the sensitivity was 90% when the analysis was performed using the NGS index, which is a combination of the diversity index and the OTUs (operational taxonomic units) value.ConclusionNGS analysis using the NGS index showed excellent sensitivity and specificity compared to culture tests. NGS analysis is therefore a useful modality for assessing infections in orthopedic cases.     

mNGS技术在儿童重症肺炎病原学诊断中的价值

目的探讨宏基因组二代测序(mNGS)技术在儿童重症肺炎病原学诊断中的价值。方法选取2017年12月至2020年6月武汉儿童医院收治住院治疗的219例社区获得性重症肺炎患儿临床资料,比较肺泡灌洗液mNGS检测与传统实验室病原检测对诊断儿童重症肺炎的诊断价值。结果 219例送检标本中mNGS共检出阳性病例190例(86.76%);而传统实验室病原共检出阳性病例173例(78.99%),两种检测方法比较,差异有统计学意义(P<0.05)。mNGS和传统实验室病原病原总检出率在各年龄段之间比较,差异有统计学意义(Χ²=76.488 7,P<0.05)。mNGS与传统实验室病原学检测均阳性患者163例,mNGS与传统实验室病原学检测均阴性患者22例,mNGS阳性而传统实验室病原学检测阴性患者27例,mNGS阴性而传统实验室病原学检测阳性患者7例。mNGS和传统实验室病原诊断儿童重症肺炎病原学灵敏度、特异度比较,差异有统计学意义(P<0.05)。结论二代测序技术较传统实验室病原检测可快速检测重症肺炎患儿病原学诊断,指导临床早期合理用药。     

Clinical investigational studies for validation of a next-generation sequencing in vitro diagnostic device for cystic fibrosis testing

Purpose: Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing. Methods: Two NGS assays – a Clinical Sequencing Assay (Sequencing Assay) and a 139-Variant Assay (Variant Assay) – were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to bi-directional Sanger sequencing and PCR as reference methods. For each study, positive agreement (PA), negative agreement (NA), and overall agreement (OA) were evaluated. Results: In the Accuracy Study, the Sequencing Assay achieved PA of 99.7% including the polyTG/polyT region and PA of 100% excluding the region. The Variant Assay achieved PA of 100%. NA and OA were >99.99% for both Assays. In the Reproducibility Study, the Sequencing Assay achieved PA of 99.2%; NA and OA were both 99.7%. The Variant Assay achieved PA of 99.8%; NA and OA were both 99.9%. Sample pass rates were 99.7% in both studies for both assays. Conclusion: This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. These NGS-based Assays had accurate and reproducible results which were comparable to or better than other methods currently in clinical use for clinical genetic testing of cystic fibrosis.     

Next-generation sequencing and its applications in molecular diagnostics

DNA sequencing is a powerful approach for decoding a number of human diseases, including cancers. The advent of next-generation sequencing (NGS) technologies has reduced sequencing cost by orders of magnitude and significantly increased the throughput, making whole-genome sequencing a possible way for obtaining global genomic information about patients on whom clinical actions may be taken. However, the benefits offered by NGS technologies come with a number of challenges that must be adequately addressed before they can be transformed from research tools to routine clinical practices. This article provides an overview of four commonly used NGS technologies from Roche Applied Science//454 Life Sciences, Illumina, Life Technologies and Helicos Biosciences. The challenges in the analysis of NGS data and their potential applications in clinical diagnosis are also discussed.