核酸基质辅助激光解吸电离飞行时间质谱技术在结核病和非结核分枝杆菌病诊断中的临床应用专家共识

核酸基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI-TOF MS)技术在结核病与非结核分枝杆菌病病原学和耐药性诊断中的应用越来越多，为早期诊断、鉴别诊断和耐药鉴定提供了快速、准确的依据。然而，在实际应用中，临床医生对标本的选择、留取、送检时机及注意事项、报告结果的解读等的理解和掌握参差不齐，尚需规范化。本共识总结了核酸MALDI-TOF MS检测技术应用于结核病和非结核分枝杆菌病诊断的临床适应证和标本采集注意事项，介绍了如何正确解读核酸MALDI-TOF MS技术鉴定分枝杆菌菌种和耐药性的报告结果，以进一步规范核酸MALDI-TOF MS技术在结核病和非结核分枝杆菌病诊断中的临床应用，提高临床诊断水平，指导临床开展早期精准有效的治疗。     

基质辅助激光解析电离飞行时间质谱快速鉴定沙门菌临床分离株

目的评估基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)在沙门菌鉴定中的临床应用,并研究其影响因素。方法用传统血清学方法鉴定沙门菌的血清型,用MALDI-TOF MS对哥伦比亚血平板、HE平板、SS平板和麦康凯平板分别培养24h、72h、120h和168h的179株沙门菌进行鉴定。结果 179株沙门菌可分为38种血清型,肠炎沙门菌(21.2%)、斯坦利沙门菌(17.3%)和Ⅰ4,5,12:i:-沙门菌(16.2%)居前3位。所有菌株在血平板、HE平板、SS平板和麦康凯平板分别培养24h和72h均能被MALDI-TOF MS准确鉴定为沙门菌。MALDI-TOF MS的耗材成本约为微生物自动生化鉴定系统(Vitek 2)的1/3,检测时间约为Vitek 2的1/7。结论在血平板、HE平板、SS平板和麦康凯平板培养3d以内的沙门菌,MALDI-TOF MS均可以准确鉴定。与Vitek 2相比,MALDI-TOF MS检测的速度更快,消耗的成本更低,并且可以直接鉴定生长在选择性培养基的沙门菌。     

从《2020年全球结核病报告》看我国结核病防治工作

《2020年全球结核病报告》从实现全球结核病防治目标的进展、新型冠状病毒肺炎对全球结核病防控的影响、全球结核病疫情的现状、全球结核病的诊断和治疗现状、全球结核病的预防、结核病筹资、结核病的研究和创新等7个方面进行了阐述,本研究系统解读报告的要点内容,并结合我国结核病防治的相关领域进展情况进行分析,为指导我国下一步结核病...     

精进臻善 惟实励新:世界卫生组织《2022年全球结核病报告》解读

结核病防控工作亟待创新性的研究与发展，以加快实现终止结核病策略。2022年10月27日，世界卫生组织发布了《2022年全球结核病报告》,对全球结核病领域的研究和创新进展进行更新。笔者就报告中结核病研究领域新诊断技术、新药与新方案，以及新疫苗等研究及创新要点进行解读，并结合我国相关领域的研究进展进行分析，以期为我国结核病防治领域同行掌握结核病研究动态，进一步开展原创性科研和创新提供参考。     

结核病的研究与创新——2021年世界卫生组织全球结核病报告解读

结核病的科研与创新是实现世界卫生组织终止结核病策略目标的三大支柱之一。2021年10月14日,世界卫生组织发布了《2021年全球结核病报告》,对全球结核病科研创新进展进行了更新。笔者就全球报告中结核领域新药、新诊断技术及新疫苗等研究及创新要点进行综述,并结合我国相关领域的研究进展进行分析,以期为我国结核病领域同行掌握结核病研究动态、进一步开展原创科研和创新提供参考。     

首届大规模伞国性耐药结核病学术大会召开

首届大规模全国耐药结核病学术大会于2009年6月13日在广州市召开。会议主题为耐药结核病诊治进展，涵盖耐药结核病的临床诊治、基础研究、预防控制等领域，国内知名结核病学专家和学者分别就本领域最新进展以专题讲座和学术讨论形式进行了交流，来自全国各省市共计248人参加了本次会议。     

基质辅助激光解吸电离飞行时间质谱在临床微生物鉴定中的应用

基质辅助激光解吸/电离飞行时间质谱(Matrix-assisted laser desorption /ionization time-of-flight mass spectrometry,MALDI-TOF MS)技术是近年发展起来的一种软电离新型有机质谱,它可以在几分钟内直接对平皿中或标本中的菌落进行鉴定。这种新而简单的方法大大降低了耗材成本和鉴定诊断时间。可靠性和准确性已在许多研究报告中得以证明,不同的系统设备也已在市场销售,在不久的将来,该项技术会在实际应用中拥有更加广阔的前景。MALDI-TOF MS将会成为替代传统人类病原体微生物鉴定手段的革新技术。本文将回顾MALDI-TOF MS的原理、发展、应用情况、与传统方法相比的优势及现阶段存在的局限性,着重强调该项技术在微生物系统的应用特点。     

基质辅助激光解吸电离飞行时间质谱在幽门螺杆菌检测中的应用

目的 建立并评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对幽门螺杆菌(Hp)的鉴定能力及Hp耐药性菌株对检测结果的影响.方法 MALDI-TOF MS自建库中的Hp来源于样本库中经传统方法鉴定和药物敏感试验并已进行全基因测序确认的菌株,随机取其中对药物全敏感的Hp菌株10株,用MALDI-TOF M...     

VITEK MALDI-TOF MS技术在临床分离诺卡菌快速鉴定中的简易流程优化

目的 本研究利用基质辅助激光解吸电离飞行时间质谱-VITEK MALDI-TOF MS,对临床分离诺卡菌进行快速、准确、简易的菌种鉴定。方法 对46株临床分离的诺卡菌进行研究,以16S rRNA和gyrB基因测序结果为参考标准,探索VITEK MALDI-TOF MS技术在诺卡菌菌种鉴定程序中的关键步骤,优化鉴定流程。结果 46株诺卡菌共有43(93.5%)株可鉴定至种水平,45(97.8%)株可鉴定至属水平。对分离率最高的盖尔森基兴诺卡菌和鼻疽诺卡菌的鉴定率更是高达100.0%(22/22和7/7株)。结论 实验结果表明,VITEK MALDI-TOF MS技术可以实现对临床分离诺卡菌的快速、准确鉴定,简易鉴定流程为广泛应用于临床微生物实验室提供了重要基础保障。     

世界卫生组织《2023年全球结核病报告》解读

2023年11月7日,世界卫生组织发布了《2023年全球结核病报告》,该报告围绕全球结核病疫情负担、登记报告、治疗转归、创新进展等内容展示了全球结核病防控工作的基本要点。作者通过对该报告的全球结核病负担、诊疗现状等方面进行解读,为结核病领域工作者全面了解全球结核病疫情现状、防控策略、行动及进展提供参考。     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of bacteria and yeasts in a clinical microbiological laboratory: a review

Recently, MALDI-TOF MS devices designed for use in clinical laboratories have been commercially introduced in various large centres worldwide. All published studies conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria and yeasts in a clinical microbiological laboratory. Although all data show that MALDI-TOF MS correctly identifies the great majority of isolates processed routinely, it cannot yet identify every such isolate. Until today, MALDI-TOF MS is inappropriate for the identification of Shigella species, pneumococci and viridans streptococci. Database upgrades and sample enrichment are essential elements to refine the MALDI-TOF MS technique, allowing the method to increase its power. For the identification of a significant proportion of yeasts, an extraction method prior to analysis in the mass spectrometer is mandatory to obtain appropriate spectra. Because of the low marginal costs, and the extreme speed of MALDI-TOF MS, the technique can improve laboratory efficiency when used early in identification protocols. Lengthier, more labour-intensive, and costlier techniques can be reserved for the minority of isolates not identified with high confidence by MALDI-TOF MS. MALDI-TOF MS also has the potential to directly identify pathogens in biological fluids, such as urine samples and blood cultures. For this application however, further well-designed prospective studies are warranted. The potential for identification at the serotype or strain level, and antibiotic resistance profiling within minutes make MALDI-TOF mass spectrometry an ongoing revolution in the clinical microbiology laboratory.     

基质辅助激光解吸电离飞行时间质谱技术对分枝杆菌液体培养系统菌种鉴定的临床研究

目的 应用基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF MS)对Bactec^TM MGIT^TM 960液体培养出的阳性培养物进行高通量、快速、准确的菌种鉴定,为临床早期、精准治疗提供病原学依据。方法 选取我院Bactec^TM MGIT^TM 960分枝杆菌快速液体培养的阳性培养物为研究对象,通过不同的孵育时间、不同大小的研磨珠处理、不同的破壁方式摸索出MALDI-TOF MS对Bactec^TM MGIT^TM 960阳性培养物鉴定的“最优处理方法”,并将所有结果与基因芯片法菌种鉴定做参照进行比对,鉴定不一致的结果通过基因测序确认。结果 Bactec^TM MGIT^TM 960液体培养报阳性后增加孵育时间可提高检出率及鉴定分值,同时使用0.5 mm的氧化锆珠研磨,使用超声震荡混匀处理,鉴定效果最佳。鉴定结果方面,液体系统报阳的420份阳性标本,基因芯片法鉴定结果为结核分枝杆菌占69.05%(290/420),非结核菌占28.81% (121/420),9例未鉴定出,未鉴定占2.14%(9/420),错误鉴定占0.95%(4/420),总鉴定率为97.86%(411/420);采用MALDI-TOF MS质谱鉴定420份阳性标本中鉴定出419例,其中结核分枝杆菌占69.05%(290/420),非结核占30.00%(126/420),星型奴卡菌0.48%(2/420),巴西奴卡菌占0.24%(1/420),1例未鉴定出,总鉴定率为99.76%。质谱鉴定分值≥2.0的400例占95.24%,分值在1.7~2.0的19例占4.52%,分值<1.7的1例占0.24%。结论 本实验实现了Bactec^TM MGIT^TM 960液体培养阳性培养物的直接质谱鉴定,缩短了培养鉴定的时间,将分枝杆菌鉴定到种水平,能区分出结核菌与非结核菌,同时也能将与分枝杆菌相似的奴卡菌鉴定出来,对临床疾病的鉴别诊断起到了积极的作用。     

核酸质谱检测结核分枝杆菌耐药方法的建立

目的建立核酸飞行质谱(MassARRAY)方法检测结核耐药基因突变检测体系,并评估其检测临床耐药结核病的应用价值。方法收集2016年1月-2019年6日上海市嘉定区中心医院肺科肺结核有治疗经历患者的菌培养样本55例,其中药物敏感样本10例,45例耐多药样本中,耐异烟肼、耐氟喹诺酮类(左氧氟沙星、莫西沙星)的样本45例、耐二线类注射药物(阿米卡星、卷曲霉素)的样本23例;供试样本均提取核酸,先用一代测序验证质谱检测的准确性,再根据临床药敏试验结果,验证质谱检测复治样本的有效性,主要评价指标为检测的灵敏度、特异性和一致性。结果与一代测序相比,该体系检测临床菌株异烟肼、氟喹诺酮类药物和二线注射类药物的灵敏度分别为100%(37/37)、100%(34/34)和100%(10/10),特异性分别为100%(18/18)、100%(21/21)和100%(45/45),总一致率100%,一致性检验Kappa值为1。以最低抑菌浓度法(MIC)为金标准,该体系检测临床菌株异烟肼、氟喹诺酮类药物和二线注射类药物的灵敏度分别为82.2%(37/45)、75.6%(34/45)和45.5%(10/22),特异性分别为100%(10/10)、100%(10/10)和100%(33/33),两种方法的总一致率为89.1%,一致性检验Kappa值为0.703。结论质谱检测基因突变与最低抑菌浓度法检测结核菌耐药总体具有一定的一致性,可快速检测结核分枝杆菌的耐药性,有望成为复治患者辅助临床治疗的新手段。     

Laboratory Biosafety: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification of security-sensitive bacteria: Considerations for Canadian Bruker users

BackgroundThe use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) systems for bacterial identification has rapidly become a front line tool for diagnostic laboratories, superseding classical microbiological methods that previously triggered the identification of higher risk pathogens. Unknown Risk Group 3 isolates have been misidentified as less pathogenic species due to spectral library availability, content and quality. Consequently, exposure to higher risk pathogens has been reported within Canadian laboratory staff following the implementation of MALDI-TOF MS. This overview aims to communicate the potential risk to laboratory staff of inaccurate identification of security-sensitive biological agents (SSBA) bacteria and to provide suggestions to mitigate.MethodsCultures were manipulated in a Biosafety Level 3 laboratory, prepared for MALDI-TOF MS analysis via full chemical extraction and analysed on a Bruker Microflex LT instrument. Data were analyzed with Biotyper software; comparing raw spectra against MS profiles in three libraries: Bruker Taxonomy; Bruker Security-Restricted; and National Microbiology Laboratory (NML) SSBA libraries. Four years of Bruker MALDI-TOF MS data acquired in-house were reviewed.ResultsIn general, the Bruker MS spectral libraries were less successful in identifying the SSBA bacteria. More successful was the NML library. For example, using a high score cut-off (greater than 2.0), the Bruker SR library was unable to identify 52.8% of our Risk Group 3 agents and near neighbours to the species-level with confidence, whereas the custom NML library was unable to identify only 20.3% of the samples.ConclusionThe last four years of data demonstrated both the importance of library selection and the limitations of the various spectral libraries. Enhanced standard operating procedures are advised to reduce laboratory exposure to SSBAs when using MALDI-TOF MS as a front line identification tool.     

Use of MALDI-TOF MS (Bruker Daltonics) for identification of Mycobacterium species isolated directly from liquid medium

ObjectivesThe aim of this study was to describe the evaluation of the use of MALDI-TOF MS for the identification of non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis directly from liquid MGIT cultures from January 2017 to December 2017.Material/methodsA total of 155 isolates (mainly respiratory) were analyzed by MALDI-TOF MS (Bruker Daltonics) directly from MGIT liquid medium with a previous extraction procedure.ResultsMALDI-TOF MS generated acceptable scores for 152 isolates (98.06%). Fifty isolates were identified as M. tuberculosis complex and the remaining 105 as NTM (M. abscessus subsp. abscessus, M. avium, M. celatum, M chelonae, M. chimaera, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. lentiflavum, M. mageritense, M. mucogenicum and M. xenopi).ConclusionsThese results indicate that MALDI-TOF MS can be useful to identify mycobacteria directly from MGIT cultures and is an accurate, rapid and cost-effective system to be used as a routine method.     

质谱技术在病原微生物诊断中的应用

质谱技术因其高灵敏度、特异性、自动化和高通量等特点,被广泛用于病原微生物蛋白质、多肽等的研究,为病原微生物诊断提供了新技术。本文以基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)和电喷雾(ESI)三重四极杆多反应监测(MRM)质谱技术在微生物诊断中的研究进展进行系统综述。     

Comparative study of MALDI-TOF MS and VITEK 2 in bacteria identification

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. This study aimed to evaluate the accuracy of MALDI-TOF MS in clinical microbiology diagnosis by comparing it with commonly-used VITEK 2 or gene sequencing.

Methods

The performances of MALDI-TOF MS and VITEK 2 were compared retrospectively for identifying routine isolates. Discrepancies were analyzed by gene sequencing analysis of the 16S genes.

Results

For 1,025 isolates, classified as 55 species of 25 genera, 1,021 (99.60%) isolates were accurately identified at the genus level, and 957 (93.37%) isolates at the species level by using MALDI-TOF MS. A total of 949 (92.59%) isolates were completely matched by both methods. Both methods found 76 unmatched isolates among which one strain had no definite identification by MALDI-TOF MS and VITEK 2 respectively. However, MALDI-TOF MS made no errors at the genus level while VITEK 2 made 6 (0.58%) errors at the genus level. At the species level, the identification error rates for MALDI-TOF MS and VITEK 2 were 5.56% and 6.24%, respectively.

Conclusions

With a lower identification error rate, MALDI-TOF MS has better performance than VITEK 2 in identifying bacteria found routinely in the clinical laboratory. It is a quick and cost-effective technique, and has the potential to replace conventional phenotype methods in identifying common bacterial isolates in clinical microbiology laboratories.     

Study on the quality of recombinant proteins using matrix-assisted laser desorption ionization time of flight mass spectrometry

Subjecxtheadingsrecombinantproteins;molecularweight;flightmassspectrometry;erythroPOietintrypticdigestsINTRODUCTIONSinceHillenkampetal[ljfirstintroducedmatrixassistedlaserdesorption/ionizationtimeofflightmassspectrometry(MALDI-TOFMS)toanalyzeproteinswithmolecularmassesgreaterthanM.10000,~DI--TOFMShasbeenwidelyusedtostudydifferentclassesofbiomoleculessuchasproteins,oligonucleotides,polysaccharidesandpolymers"--".Comparedtothetraditionaltechniques,suchassodiumdodecylsulfatepolyacryl…     

分枝杆菌的实验室检查与质量控制

我国结核病有上升趋势，实验室的分枝杆菌细菌学检查就成为指导流行病学调研和结核病诊疗的重要手段，因此做好分枝杆菌细菌学检验工作，是每个实验室义不容辞的职责。但如何做好则是摆在我们面前一道非常重要的研究课题。笔者通过总结和回顾广州市胸科医院多年来的经验与教训，把在实验室工作中得出的一些心得体会撰写成文，旨在与同道们一起研究如何做好实验室的检测工作，为流行病学调研和结核病诊疗打下坚实的技术基础。     

基质辅助激光解吸电离飞行时间质谱在真菌快速鉴定中的应用

真菌是广泛分布于自然界的真核生物,对人类具有致病性的约300种。不同种真菌的生长特性、临床特点及耐药性不同。真菌生长缓慢,传统的培养鉴定方法所需时间较长,且日益多样化的真菌,使鉴定难度不断增加,这些均限制了临床的早期诊断和针对性治疗。基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization-time of flight mass spectrometry,MALDI-TOF MS)是一种新兴的诊断技术,可以通过直接检测生物标志物(蛋白)来鉴定病毒、细菌、分枝杆菌等,具有操作简便、快速、准确率高、成本低的特点。本文对MALDI-TOF MS在真菌鉴定中的应用进行综述,发现其对酵母样真菌的正确鉴定率可达到94%以上,丝状真菌正确鉴定率也可达到89%,可满足临床实验室鉴定真菌的需求。