宏基因组二代测序技术在慢性肺曲霉病诊断中的价值

目的：评估宏基因组二代测序技术在慢性肺曲霉病诊断中的价值。方法：回顾性收集2017年9月至2019年9月在复旦大学附属中山医院感染病科住院的疑似慢性肺曲霉病患者,并同步采集标本进行传统培养、血清烟曲霉特异性抗体IgG检测及mNGS检测,分析mNGS在慢性肺曲霉病诊断中的价值。 结果：共有78例疑似慢性肺曲霉病的患者纳入分析,根据诊断标准,最终诊断CPA 35例,non-CPA 43例。送检的的78例样本中,痰标本55例、支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)标本17例、肺组织标本6例。与最终诊断相比,mNGS诊断CPA的敏感度和特异度分别为65.7%和86.0%,烟曲霉特异性抗体IgG的敏感度和特异度分别为48.6%和90.7%,传统培养的敏感度和特异度分别为28.6%和93.0%,其中mNGS的敏感度显著高于传统培养方法(P=0.001)。对于临床诊断的CPA患者,mNGS检测的阳性率为50.0%。mNGS、烟曲霉特异性抗体IgG和传统培养方法的ROC曲线下面积(Area Under Curve, AUC)分别为0.759、0.696和0.608。在所有最终诊断为CPA的35例患者的样本中,痰标本、BALF标本和肺组织标本的mNGS阳性率分别为56.5%(13/23)、77.8%(7/9)和100%(3/3),但三组之间的差异无统计学意义。结论：在CPA的诊断中,mNGS的敏感度显著高于传统培养方法,在传统诊断方法无法确诊的患者中,mNGS仍有较高的阳性率,同时在检测时间上具有明显优势。     

血清半乳甘露聚糖抗原及烟曲霉特异性抗体检测在侵袭性肺曲霉病诊断中的价值

目的探讨血清半乳甘露聚糖(GM)抗原及烟曲霉特异性抗体检测在侵袭性肺曲霉病(IPA)临床诊断中的应用价值。方法将124例疑似IPA的住院患者分为IPA组(46例)和非IPA组(78例),检测其血清GM抗原及烟曲霉特异性IgG、IgM抗体,并对其临床资料进行回顾性分析。结果 IPA组病理确诊18例,临床诊断28例。分别以0.5、1.0 ng/mL为阈值,GM抗原诊断IPA的敏感性和特异性分别为58.7%、74.4%和50.0%、83.3%。烟曲霉特异性IgG抗体诊断IPA的敏感性和特异性分别为80.4%、91.0%,烟曲霉特异性IgM抗体诊断IPA的敏感性和特异性分别为71.7%、87.2%。IPA组烟曲霉特异性IgG、IgM抗体水平均高于非IPA组(P0.05)。结论血清GM抗原(以1.0 ng/mL为阈值)及烟曲霉特异性IgG、IgM抗体检测对于IPA的诊断均具有较高的特异性(80%),烟曲霉特异性IgG抗体检测的敏感性、特异性均优于IgM抗体。     

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。     

BALF-GM联合曲霉IgG及IgM抗体对慢性肺曲霉病的诊断价值

目的 探讨肺泡灌洗液半乳甘露聚糖（BALF-GM）联合曲霉IgG、IgM抗体检测在慢性肺曲霉病（CPA）中的诊断价值。方法 将确诊为CPA的88例患者作为研究组,疑似CPA经确诊后排除的64例患者作为对照组。回顾性分析所有患者的BALF-GM、曲霉IgG及IgM抗体水平,采用受试者工作特征（ROC）曲线分析各检测方法BALF-GM、曲霉IgG、IgM及其联合检测对CPA的诊断价值。结果 BALF-GM诊断CPA价值较高,曲霉IgG、IgM等指标及其联合检测的诊断价值较低,BALF-GM联合曲霉IgG/IgM检测灵敏度有明显提高,但特异度下降。研究组BALF-GM、曲霉IgG、IgM含量均高于对照组,2组比较差异均有统计学意义（P均< 0.05）;2组BALF-GM、曲霉IgG、IgM及联合检测阳性率比较差异均有统计学意义（P均< 0.05）。ROC曲线分析显示BALF-GM诊断CPA、BALF-GM+曲霉IgG/IgM联合诊断CPA的曲线下面积均较高,灵敏度、特异度高,曲霉IgG、IgM及IgG/IgM联合检测的诊断价值均较低。结论 BALF-GM诊断CPA具有较高的灵敏度和特异度;BALF-GM联合曲霉IgG/IgM检测可提高其诊断灵敏度,但特异度会显著降低,因此需结合BALF-GM单独检测的高特异度来尽量降低假阴性的发生。     

3种人体包虫病IgG抗体检测试剂盒的性能评价

目的对3种人体包虫病IgG抗体检测试剂盒的检验性能进行初步评价。方法收集四川大学华西医院包虫病确诊的112例患者血清标本作为包虫病组;收集60例非包虫病患者及体检者血清标本作为非包虫病组,同时用珠海海泰包虫病IgG抗体检测试剂盒(酶联免疫吸附法)、新疆贝斯明包虫病特异抗体检测试剂盒(胶体金法)、上海新吉而囊型/泡型包虫病抗体检测试剂盒(胶体金法)进行检测,计算3种试剂的灵敏度、特异度、Youden指数,评价3种试剂的灵敏度、特异度、Youden指数的差异,采用Kappa一致性检验,分析3种试剂检测结果与临床诊断的一致性。结果海泰、贝斯明、新吉而试剂盒的灵敏度分别为94.64%(106/112)、92.86%(104/112)、96.43%(108/112);特异度分别为100.00%(58/58)、91.38%(53/58)、93.10%(54/58)。3种试剂的灵敏度和特异度比较,差异无统计学意义(P0.05);Youden指数分别为0.965、0.924、0.953,Kappa值分别为0.923、0.832、0.895。结论 3种人体包虫病IgG抗体检测试剂盒均具有较好的灵敏度、特异度、准确度,与临床诊断一致性较高。     

电化学发光免疫分析法检测梅毒特异性抗体的临床应用及假阳性分析

目的 评价电化学发光免疫分析法(ECLIA)在筛查梅毒特异性抗体中的临床应用价值,并分析假阳性产生的原因。方法 选取2020年9月至2021年8月在该院就诊的门诊及住院78 374例患者标本,均采用ECLIA筛查梅毒特异性抗体,阳性标本和临界标本采用梅毒螺旋体颗粒凝集试验(TPPA)进行确证,计算ECLIA检测的灵敏度、特异度、阳性预测值和阴性预测值,并对假阳性结果进行分析。结果 ECLIA筛查梅毒特异性抗体的灵敏度、特异度、阳性预测值和阴性预测值分别为100.00%、99.94%、97.65%、100.00%;当发光比值>20.00时,ECLIA与TPPA的阳性符合率为100.00%。结论 ELCIA适合大规模的梅毒特异性抗体筛查试验,可为临床诊断梅毒提供重要血清学诊断依据。     

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。     

流行性乙型脑炎病毒IgG抗体捕获酶联免疫吸附试验检测方法的建立

目的利用流行性乙型脑炎(乙脑)病毒特异性单克隆抗体建立检测人血清中乙脑病毒IgG抗体的捕获ELISA方法。方法将乙脑病毒特异性单克隆抗体包被在固相载体上,加入灭活并经过纯化的乙脑病毒抗原,再加入待测血清,经洗涤、显色等判定最终检测结果。采用免疫荧光法作为对照。结果与免疫荧光法相比较,本方法的灵敏度为95.6%,特异度为85.7%,一致性为91.3%;本方法的灵敏度和特异度高于市售的两种检测试剂。结论本研究所建立的乙脑病毒IgG抗体捕获ELISA方法具有良好的检测性能,可以应用于正常人群血清中乙脑病毒IgG抗体的筛查。     

瓜氨酸合成蛋白抗体等三种抗体联合检测对类风湿关节炎的诊断意义

目的:探讨检测瓜氨酸合成蛋白抗体(CPA)及其联合抗环瓜氨酸肽抗体(抗CCP)和抗角蛋白抗体(AKA)的检测,在类风湿关节炎(RA)的诊断意义。方法:采用酶联免疫固相吸附试验(ELISA)和间接免疫荧光法测定65例RA患者血清中的CPA、抗环瓜氨酸肽抗体(抗CCP)和抗角蛋白抗体(AKA)水平。并以99例非RA患者以及173例健康者的CPA检测作为对照。结果:以6.25 U/ml为临界值,CPA对RA诊断的敏感性为64.6%,特异性为94.9%,阳性、阴性预测值分别为76.4%、91.3%,试验诊断准确度达88.7%。RA患者血清CPA水平与抗CCP之间无线性相关关系,抗CCP、AKA阳性率分别为67.6%、75.4%,CPA与抗CCP、AKA联合检测可提高RA阳性诊断率。骨关节侵蚀患者组与未出现骨损伤组间CPA水平没有明显差异。结论:CPA是协助诊断RA的一个新的特异性血清学指标,同时结合抗CCP抗体和AKA的检测可以提高RA阳性诊断率,适合在小规模实验室推广使用。     

血清曲霉IgG型抗体检测对器官移植术后肺曲霉感染的诊断

目的:探讨血清曲霉IgG型抗体检测在器官移植术后肺曲霉感染中的诊断价值。方法:酶免分析法检测肺曲霉感染高危患者血清中曲霉特异性IgG抗体。结果:血清曲霉IgG抗体检查的灵敏度和特异度分别为50%和100%。移植后1个月内血清曲霉IgG抗体检测的灵敏度为40%,而1个月后其灵敏度为80%。结论:特异性曲霉IgG抗体测定不能作为器官移植后肺曲霉病的早期诊断标准,但对感染后期的回顾性诊断和随访有一定意义。     

Application of metagenomic next-generation sequencing for bronchoalveolar lavage diagnostics in patients with lower respiratory tract infections

ObjectiveMetagenomic next-generation sequencing (mNGS) has the potential to overcome the shortcomings of traditional culture methods. This study aimed to assess the diagnostic value of mNGS in patients with lower respiratory tract infections (LRTIs).MethodsThis retrospective observational study sequentially enrolled 47 patients with LRTIs admitted to Shenzhen Hospital of Southern Medical University between February 2019 and November 2020. Pathogens in bronchoalveolar lavage fluid (BALF) samples were investigated to compare diagnoses by mNGS with culture methods.ResultsCompared with culture methods, mNGS had a diagnostic sensitivity of 80% and a specificity of 35.13% with an agreement rate of 44.68% between these two methods. mNGS significantly increased the pathogen detection rate.ConclusionsmNGS may show some advantages in identifying a wide range of LRTI pathogens, improving the sensitivity for viruses and atypical pathogens. The clinical application of NGS technology is worth looking forward to.     

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。     

Potential of metagenomic next-generation sequencing in detecting infections of ICU patients

BackgroundDue to the limitations of traditional microbiological detection techniques in evaluating complicated infections in ICU patients, it is necessary to explore novel and effective methods to improve the clinical detection of ICU patients’ infections.ObjectiveThis study aimed to evaluate the efficiency and specificity of mNGS in screening pathogens in the blood, deep phlegm, urine, and other sample types of ICU patients exploring an effective method for infection detection.MethodsA total of 56 ICU patients with 131 samples were included in this study. The sample types included blood, deep phlegm, urine, drainage, anal swabs, and other types. Samples were analyzed by both conventional detection method and mNGS tests. The diagnosis efficiency and consistency of the two methods were compared. The distribution of the identified pathogens was analyzed. Moreover, the clinical features of patients with mNGS-positive or mNGS-negative results were compared.ResultsThe positive rate of mNGS was 81.7% (107/131) including 3.1% (4/131) weakly positive, while the positive rate of traditional detection was only 30.5%, including 29 strong positive results and 11 weak positive results. Additionally, there were 41 patients chose to adjust anti-infection strategies according to the results of mNGS, which significantly saved treatment costs. The mNGS-positive patients showed a shorter ICU hospitalization and higher intention to adjust anti-infection strategies than the mNGS-negative patients.ConclusionmNGS is of great potential for the pathogen detection of ICU patients, and has a higher detection rate than traditional detection methods. Further clinical application investigations can be carried out to expand the application of mNGS.     

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.     

Metagenomic next-generation sequencing for detection of pathogens in children with hematological diseases complicated with infection

ObjectiveInfection is one of the most common causes of death in children with hematological diseases. Here, we aim to investigate the value of metagenomic next-generation sequencing (mNGS) in the detection of causative pathogens in children with hematological diseases.MethodsIn this retrospective study, specimens from children with hematological diseases, who were admitted to Sun Yat-Sen University between June 2019 and September 2021, were collected for culture and mNGS.ResultsA total of 67 pediatric patients were enrolled, and 96 specimens were collected. The positive rate of mNGS was significantly higher than that of culture (57.2% vs 12.5%, P < 0.01). The concordance (90.9%, 10/11) between the positive results of the two methods was high. mNGS detected more cases with Pneumocystis jeroveci, Aspergillus flavus, viruses, and some rare pathogens than culture. Mixed infections were detected by mNGS in 16 cases. Clinical anti-infective treatment was adjusted according to the results of mNGS, the conditions of most patients improved.ConclusionCompared to culture, mNGS shows great advantages in diagnosing bacterial, fungal, viral, and mixed infections in children with hematologic diseases, positively impacting clinical care. mNGS can be used as a complement to culture for pathogen detection.