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脊柱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)技术对脊柱感染病原学诊断的价值。方法:回顾性分析2018年1月至2019年12月复旦大学附属中山医院收治的24例疑似脊柱感染患者的病例资料,采用常规微生物培养法和mNGS技术检测脊柱组织标本。结果:mNGS总体检测阳性率高于常规培养法(62.5%vs 35.0%),mNGS平均耗时36～48 h,明显短于常规培养法的平均天数21.8 d。脊柱感染主要检出病原体为结核分枝杆菌、金黄色葡萄球菌、大肠埃希菌。结论:mNGS能够快速检测脊柱感染病原体,为临床早期精准治疗提供重要的病原学依据。     

脑脊液宏基因组二代测序对结核性脑膜炎诊断价值

目的 分析比较结核性脑膜炎患者应用宏基因组二代测序(metagenomic next-generation sequencing, mNGS)、脑脊液抗酸染色、结核分枝杆菌培养、结核分枝杆菌/利福平耐药实时荧光定量核酸扩增(Xpert MTB/RIF)检测结核分枝杆菌的结果,探讨mNGS对结核性脑膜炎的诊断价值。方法 临床确诊的结核性脑膜炎患者34例,收集患者临床特征及脑脊液mNGS、常规检测、生化检测、抗酸染色、结核分枝杆菌培养、Xpert MTB/RIF检测等结果,比较脑脊液mNGS与病原学检测结核分枝杆菌检出率。结果 34例结核性脑膜炎患者发热29例,头痛27例,颈项强直14例,精神行为异常9例,意识障碍11例,认知功能障碍4例,继发性癫痫5例。脑脊液压力升高26例[240(150,330)mm H₂O],白细胞计数升高32例[194.0(92.5,453.5)×10⁶/L],均以单核细胞为主,蛋白质水平升高32例[1.58(0.95,2.57)g/L],葡萄糖水平降低20例[1.93(1.19,2.95)mmol/L],氯离子水平降低...     

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...     

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。     

宏基因组二代测序在涂阴性肺结核中的应用进展

结核病是全球关注的公共卫生问题,全球结核病、耐药结核病以及结核病死亡患者数量都在逐步增长,我国结核病形势也不容乐观。早期准确的病原学诊断是有效治疗的关键,但我国大多数的结核患者病原学检测阴性或缺乏病原学检测结果。对于病原学阴性肺结核,目前常用病原学检测方法尚不能满足临床的诊断需求。宏基因组二代测序（m NGS）的出现颠覆了传统的结核杆菌检测技术以及病原学诊断方法,为涂阴性肺结核的早期诊断、早期治疗提供了可靠参考。本文就现有结核分枝杆菌（MTB）检测方法及国内外开展的m NGS技术的应用作一个综述,重点分析m NGS在涂阴性肺结核诊断中的优势与局限性,为临床有效诊断涂阴性肺结核及合理应用m NGS提供参考。     

宏基因组二代测序在重症肺炎患者病原学中的应用

目的 探究重症肺炎患者肺泡灌洗液（BALF）宏基因组二代测序（mNGS）与传统培养病原学诊断的差异以及mNGS结果对抗生素方案调整的影响。方法 回顾性分析2019年8月至2021年4月于贵州医科大学附属医院重症医学科住院期间,同时送检BALF传统培养和mNGS的重症肺炎患者。比较两种病原学检测结果的差异,分析mNGS与传统培养诊断的阳性率、一致性及抗生素方案的调整。结果研究期间共纳入患者75例,其中mNGS的阳性率为97.3%,传统培养阳性率为41.3%;mNGS细菌的检出率为94.7%,传统培养细菌检出率为38.7%;mNGS真菌检出次数最多的为耶氏肺孢子菌24.0%。mNGS对于罕见菌、不典型菌、真菌以及病毒的阳性率远高于传统培养。两组病原学检测结果一致性差（Kappa=0.038,P=0.229）,mNGS较传统培养检出更多种类的微生物。基于mNGS检测结果,临床医生对于69%的患者进行抗生素方案的调整。结论 mNGS在重症肺炎患者病原学诊断中敏感性高,对于免疫抑制重症肺炎患者尤为适用。同时,mNGS可以指导临床医师调整抗生素策略。     

宏基因二代测序在开颅术后中枢神经系统感染诊断中的应用

目的 评估宏基因二代测序(metagenomic next generation sequencing,mNGS)在开颅术后中枢神经系统感染诊断中的应用.方法 纳入2020年4月至2021年4月福建医科大学附属泉州第一医院共48例开颅术后疑似中枢神经系统感染的患者,即时留取脑脊液.所有患者均完善脑脊液常规检查、生化检查、细菌培养和mNGS,待全部患者明确是否合并中枢神经系统感染后回顾性评估细菌培养方法、mNGS对中枢神经系统感染诊断的效果.结果 共31例患者临床诊断颅内感染,其中23例脑脊液mNGS测序阳性,细菌培养阳性10例.两种检验方法Kappa值为0.375、P=0.002.mNGS检测方法诊断效能高于细菌培养:脑脊液mNGS测序诊断术后颅内感染的灵敏度74.2％、特异度94.1％、阳性预测值95.8％、阴性预测值66.7％、约登指数68.3、曲线下面积0.842(95％CI:0.725～0.958,P<0.05);细菌培养的灵敏度、特异度、阳性预测值、阴性预测值、约登指数、曲线下面积分别为32.3％、94.1％、90.9％、43.2％、26.4、0.632(95％CI:0.474～0.790,P=0.134);两种检测方法曲线下面积经过Delong检验,结果显示差异有统计学意义(Z=4.139,P<0.05).结论 mNGS与细菌培养检验手段相比,有助于开颅术后中枢神经系统感染早期诊断.     

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。     

Clinical metagenomic sequencing for rapid diagnosis of pneumonia and meningitis caused by Chlamydia psittaci

BACKGROUNDChlamydia psittaci (C. psittaci) is a gram-negative intracellular parasitic pathogenic bacterium that can infect avian and mammalian hosts, including humans. The detection of C. psittaci infections typically relies on traditional antigen-based immunoassays or serological testing that often lack sensitivity and/or specificity. Metagenomic next generation sequencing (mNGS) is an emerging tool for diagnosis.AIMTo demonstrate that mNGS represents a valuable tool for rapid, sensitive, and accurate pathogen detection including C. psittaci infections.METHODSFour cases of psittacosis pneumonia and one case of pediatric psittacosis meningitis were diagnosed between December 2019 and May 2020 using mNGS at Changzhou Second People’s Hospital affiliated to Nanjing Medical University. Patients’ clinical characteristics, manifestations, and treatment histories were retrospectively evaluated. RESULTSAll five patients had a history of exposure to wild (psittacine or other birds) or domesticated birds (chickens). All patients had a high fever (> 39℃) and three of them (60%) experienced organ insufficiency during the disease. The laboratory data showed normal to slightly increased leucocyte and neutrophil counts, and elevated procalcitonin levels in all five cases, and very high C-reactive protein levels in psittacosis pneumonia patients. mNGS identified a potential pathogen, C. psittaci, in patients’ bronchoalveolar lavage fluid or cerebrospinal fluid. Computed tomography revealed lung air-space consolidation, pleural thickening, and effusion fluid buildup in psittacosis pneumonia cases, and an arachnoid cyst in the right temporal lobe of the pediatric psittacosis meningitis patient. All patients experienced complete recovery following the administration of targeted anti-chlamydia therapy. CONCLUSIONThis study not only demonstrated that mNGS represents a valuable tool for rapid, sensitive, and accurate pathogen detection, but also raised public health concerns over C. psittaci infections.     

Clinical application of metagenomic next‐generation sequencing technology in the diagnosis and treatment of pulmonary infection pathogens: A prospective single‐center study of 138 patients

IntroductionRapid and accurate pathogen identification is essential for the treatment of pneumonia. Metagenomic next‐generation sequencing (mNGS) is a newly developed technology to obtain microbial nucleic acid sequence information quickly, efficiently, and without bias.MethodsWe performed shotgun metagenomic next‐generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) for pathogen identification in pneumonia in a prospective study with 138 patients from a single center. We compared the results of mNGS with standard methods including culture, staining, and targeted PCR and evaluated the clinical applicability of mNGS.ResultsMost of the patients (128/138, 92.75%) were cured or improved. One patient (1/138, 0.72%) died because of acute gastrointestinal bleeding, and 9 patients (9/138, 6.52%) showed no improvement. mNGS identified more bacteria (53 versus 27), fewer fungi (8 versus 31), and more viruses (16 versus 1) than standard methods. In total, treatment in 34 out of 138 cases (24.64%) was adjusted and optimized because of mNGS results. Positive mNGS results contributed to a definitive diagnosis in 23 cases (16.67%), which helped guide treatment decision by either adjusting the antibiotics without de‐escalation or continuing the empirical treatment. mNGS also confirmed no active infection in 11 cases (7.97%) allowed for antibiotic de‐escalation.ConclusionThis prospective clinical study evaluated the clinical utility of mNGS for the diagnosis of pneumonia and showed that mNGS of BALF provides valuable information for effective treatment.     

Comparative diagnostic utility of metagenomic next‐generation sequencing,GeneXpert, modified Ziehl–Neelsen staining,and culture using cerebrospinal fluid for tuberculous meningitis: A multi‐center,retrospective study in China

BackgroundEarly diagnosis of tuberculosis meningitis (TBM) remains a great challenge during clinical practice. The diagnostic efficacies of cerebrospinal fluid (CSF)‐based mycobacterial growth indicator tube (MGIT) culture, modified Ziehl–Neelsen (ZN) staining, Xpert MTB/RIF, and metagenomic next‐generation sequencing (mNGS) for TBM remained elusive.MethodsA total of 216 adult patients with suspicious TBM were retrospectively enrolled in this multi‐cohort study. The diagnostic performances for MGIT, modified ZN staining, Xpert MTB/RIF, and mNGS using CSF samples were evaluated.ResultsUniform clinical case definition classified 88 (40.7%) out of 216 patients as the definite TBM, 5 (2.3%) patients as probable TBM cases, and 24 (11.1%) patients as possible TBM cases. The sensitivities of MGIT, modified ZN staining, Xpert MTB/RIF, and mNGS for TBM diagnosis against consensus uniform case definition for definite TBM were 25.0%, 76.1%, 73.9%, and 84.1%, respectively. Negative predictive values (NPVs) were 66.0%, 85.9%, 84.8%, and 90.1%, respectively. The sensitivities of MGIT, modified ZN staining, Xpert MTB/RIF, and mNGS for TBM diagnosis against consensus uniform case definition for definite, probable, and possible TBM were 18.8%, 57.3%, 55.5%, and 63.2%, respectively. Negative predictive values (NPVs) were 51.0%, 66.4%, 65.6%, and 69.7%, respectively. mNGS combined with modified ZN stain and Xpert could cover TBM cases against a composite microbiological reference standard, yielding 100% specificity and 100% NPV.ConclusionMetagenomic next‐generation sequencing detected TBM with higher sensitivity than Xpert, ZN staining and MGIT culture, but mNGS cannot be used as a rule‐out test. mNGS combined with Xpert or modified ZN staining could enhance the sensitivity of diagnostic tests for TBM.     

宏基因二代测序技术对医院传染性疾病防控的价值

目的:探讨宏基因二代测序(metagenomic next-generation sequencing, mNGS)技术对医院传染性疾病防控的价值。方法:回顾性分析2018年1月1日至2019年12月31日复旦大学附属中山医院收治的5例传染性疾病患者的病例资料,比较其常规微生物培养和mNGS结果。结果:5例患者常规微生物培养均为阴性,mNGS结果:炭疽芽孢杆菌1例,霍乱弧菌1例,结核分枝杆菌复合群1例,三日疟原虫1例,恶性疟原虫1例。根据mNGS结果,提早确诊传染性疾病并调整医院感染防控措施,随访30 d内,未出现院内传播新发病例。结论:mNGS在传染性疾病的早期识别与诊断中有重要作用,对于医院感染防控早期干预起到良好的指导效果。     

宏基因二代测序技术指导下的皮肤炭疽院内感染防控1例报告

炭疽是一种由炭疽芽孢杆菌引起的人兽共患病,主要以皮肤炭疽为主,属于我国法定乙类传染病。本研究报告1例33岁男性病例,自述虫咬伤后出现右前臂皮疹,自行挑破后出现发红、水泡伴少量脓液,常规微生物培养结果为阴性,血宏基因二代测序(metagenomic next-generation sequencing,mNGS)结果示炭疽芽孢杆菌。本研究从早期诊断和指导院内感染防控角度,探讨mNGS的临床应用价值。     

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。     

宏基因二代测序技术辅助诊断波氏假阿利什霉肺部感染1例报告

假阿利什霉属肺部感染影像学表现与曲霉属感染无明显差异,但药物敏感性不同,因此菌种鉴定对指导临床治疗意义重大。本研究报告1例有哮喘病史的老年女性病例,出现咳嗽、咳痰伴胸闷,影像学表现为肺部空洞,经痰微生物培养和宏基因二代测序技术诊断为波氏假阿利什霉感染。国内外尚无宏基因二代测序技术诊断此疾病相关报道,该病例旨在为其在肺部感染的诊治提供参考。     

一例染色体微缺失和微重复患儿的细胞遗传学及分子生物学分析

目的 确定1例畸形儿的核型,探讨二代测序(NGS)技术在分子细胞遗传学中的应用。方法 对1例畸形儿、其父母及祖父三代人行外周血染色体G显带核型分析、NGS检测。结果 G显带染色体分析显示患儿、其父母及祖父核型均未见异常; NGS结果显示患儿46,dup(X)(q27.2q28); dup(Y)(p11.2p11.31); del(Y)(q11.223q11.23); del(9)(p23p24.3),其父母及祖父结果均未见异常。结论 通过NGS确定染色体微缺失及微重复是导致患儿多发畸形的主要原因。