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。     

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。     

飞行时间质谱技术鉴定丝状真菌的研究进展

侵袭性真菌感染与免疫力低下患者的高发病率和高病死率密切相关,其早期诊治对提高患者救治率至关重要。基质辅助激光解析电离飞行时间质谱(matrix-assisted laser-desorption/ionization time of flight mass spectrometry,MALDI-TOF MS)技术通过检测真菌中固有的特异性蛋白从而实现对丝状真菌快速、准确的鉴定。本文从技术原理、标本前处理、鉴定效果及影响因素等方面对MALDI-TOF MS技术鉴定丝状真菌的研究进展作一综述。     

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。     

Bruker Biotyper和Vitek MS系统质谱分析鉴定革兰阴性菌临床分离株的比较研究

目的比较并评价两种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)系统——Bruker MALDI Biotyper系统(以下简称Bruker Biotyper系统)和MALDITOF Vitek MS系统(以下简称Vitek MS系统)在革兰阴性菌临床分离株鉴定中的应用。方法收集沈阳军区总医院2012年3月—2013年1月分离自血液、尿液、脑脊液、分泌物、伤口拭子和痰液临床标本的革兰阴性菌共120株。应用Vitek 2 Compact生化鉴定系统将每株细菌鉴定到种,而后采用Bruker Biotyper和Vitek MS系统对其进行鉴定,三者鉴定结果存在差异的菌株经16S rDNA测序最终确认菌种。结果对于120株革兰阴性菌临床分离株,Bruker Biotyper和Vitek MS系统在属的水平上正确鉴定率分别为95.0%和92.5%,在种的水平上正确鉴定率分别为89.2%和86.7%,差异均无统计学意义。结论 Bruker Biotyper和Vitek MS系统对革兰阴性菌临床分离株的正确鉴定率不存在差异,两种系统的鉴定结果与各自的图谱数据库有密切关系。     

基于MALDI-TOF-MS和MLVA技术对军团菌的鉴定和分型研究北大核心CSCD

目的了解福建省4个地区军团菌的基因特征与分布情况。方法采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)对2013-2020年分离自福建省4个城市的32株9个血清型(Lp1、Lp3、Lp4、Lp5、Lp6、Lp7、Lp8、Lp9、L. micdadei)军团菌进行鉴定,结合多位点串联重复序列分析(MLVA)检测分析其中8个位点(lpsm1,lpsm3,lpsm13,lpsm17,lpsm19,lpsm33,lpsm34,lpsm35)对其中的13株Lp1进行分型。结果 32株军团菌均可通过MALDI-TOF MS检测到属种水平,鉴定分值平均值为2.04±0.16;通过蛋白聚类可以分为A、B、C 3个群;对其中的13株Lp1菌株进行MLVA分型,菌株间相似度为40.9%~87.5%,按照100%的相似水平可分为13个MLVA型,无优势MLVA型。Lp1 2013-25(7号菌)和Lp1 2013-14(9号菌)在MALDI-TOF MS和MLVA两种聚类均显示亲缘关系较近,并与其他菌株均存在差异。结论 MALDI-TOF MS技术可应用于军团菌的快速鉴定,但MALDI-TOF MS与MLVA用于军团菌的分子分型并不完全一致,可联合应用分析军团菌的群体遗传关系。     

慢性外耳道炎患者分离溶藻弧菌的鉴定及生物学特性分析

目的探讨溶藻弧菌的鉴定方法和生物学特性,为溶藻弧菌感染的诊断提供依据。方法对1例慢性外耳道炎患者耳部脓液标本分离的病原菌通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)、API细菌生化反应鉴定卡、Vitek 2 Compact鉴定仪和16S rRNA基因测序进行鉴定,采用抗菌药物敏感性试验检测耐药表型。结果该菌株鉴定为溶藻弧菌,药敏试验显示其对哌拉西林、氨苄西林/舒巴坦、哌拉西林/他唑巴坦、头孢他啶、头孢吡肟、亚胺培南、美罗培南、庆大霉素、阿米卡星、左氧氟沙星、环丙沙星和甲氧苄啶/磺胺甲噁唑敏感,对氨苄西林耐药,与患者的临床诊断及疾病预后相符合。结论 MALDI-TOF MS和16S rRNA基因测序对溶藻弧菌的鉴定准确可靠,对溶藻弧菌感染诊断具有重要价值,可供临床参考。     

质谱仪在真菌鉴定中的应用与评价

目的探讨质谱仪在真菌感染中的应用,评价其鉴定真菌的能力。方法选取分离自我院住院患者的真菌菌株327株,用质谱仪进行快速鉴定,并与VITEK-2(酵母样真菌)和显微镜检查(丝状真菌)的鉴定结果进行比对,差异结果用分子生物学方法确认鉴定。结果依照质谱仪评分标准,227株酵母样真菌在种水平(2.0)的鉴定率为90.31%,在属水平(1.7)为98.68%;丝状真菌在种水平的鉴定率为74.00%,在属水平为94.00%。对临床常见的曲霉菌鉴定正确率可达96.74%。结论质谱仪在鉴定真菌的种属水平上都达到了令人满意的结果,尤其鉴定酵母菌和曲霉菌的能力更为突出,可作为临床实验室的常规检测方法。     

The Ongoing Revolution of MALDI-TOF Mass Spectrometry for Microbiology Reaches Tropical Africa

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) represents a revolution in routine pathogen identification in clinical microbiology laboratories. A MALDI-TOF MS was introduced to tropical Africa in the clinical microbiology laboratory of the Hôpital Principal de Dakar (Senegal) and used for routine pathogen identification. Using MS, 2,429 bacteria and fungi isolated from patients were directly assayed, leading to the identification of 2,082 bacteria (85.7%) and 206 fungi (8.5%) at the species level, 109 bacteria (4.5%) at the genus level, and 16 bacteria (0.75%) at the family level. Sixteen isolates remained unidentified (0.75%). Escherichia coli was the most prevalent species (25.8%) followed by Klebsiella pneumoniae (14.8%), Streptococcus agalactiae (6.2%), Acinetobacter baumannii (6.1%), Pseudomonas aeruginosa (5.9%), and Staphylococcus aureus (5.9%). MALDI-TOF MS has also enabled the detection of rare bacteria and fungi. MALDI-TOF MS is a powerful tool for the identification of bacterial and fungal species involved in infectious diseases in tropical Africa.     

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.     

Eficacia de la espectrometría de masas MALDI-TOF en la identificación de bacterias anaerobias

Aim of the study

MALDI-TOF mass spectrometry (MS) is becoming a major resource in the Clinical Microbiology laboratory. Results on some groups of microorganisms are still controversial. We have studied the reliability of MALDI-TOF MS for the identification of anaerobic clinical isolates was studied compared to conventional biochemical methods, with rRNA 16S sequencing being used as a reference when discrepancies arose.

Material and methods

A total of 126 anaerobic bacteria clinical isolates were studied by using API20A kits (bioMérieux, Marcy l’Étoile, France) and MALDI-TOF MS (Autoflex II, Bruker Daltonics, Germany), and using the data library BioTyper 2.0 (Bruker Daltonics, Germany). When discrepancies arose, or MALDI-TOF MS was not able to identify any microorganism, rRNA 16S sequencing was used as the reference standard.

Results

The biochemical method and MALDI-TOF MS agreed in identifying 60.9% of isolates at species level, and 20.3% of isolates at genus level. Among the 48 discrepancies observed, rRNA 16S sequencing supported MALDI-TOF MS identification, at species level, in 32 isolates (66.7%), and in 8 isolates (16.7%) at genus level. rRNA 16S sequencing supported biochemical identification in only two isolates (4.2%) at species level, and in 26 isolates (54.2%) at genus level. The eight isolates for which MALDI-TOF MS did not manage to identify, or the identification obtained was rejected by sequencing, belonged to species that are still not added to the BioTyper II data library.

Conclusions

Results obtained in this study show that, overall, MALDI-TOF MS identification of anaerobic bacteria is more reliable than identification obtained by conventional biochemical methods (24% more correct identifications at species level). The number of major errors (incorrect identification at the genus level) is also 2.5-times lower. Moreover, all the major errors obtained by MALDI-TOF MS were due to the absence of some species in the data library. Thus, when data libraries are more complete, reliability differences between both methods will probably be even higher.     

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.     

MALDI-TOF MS versus VITEK 2 ANC card for identification of anaerobic bacteria

Background

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an accurate, rapid and inexpensive technique that has initiated a revolution in the clinical microbiology laboratory for identification of pathogens. The Vitek 2 anaerobe and Corynebacterium (ANC) identification card is a newly developed method for identification of corynebacteria and anaerobic species. The aim of this study was to evaluate the effectiveness of the ANC card and MALDI-TOF MS techniques for identification of clinical anaerobic isolates.

Methods

Five reference strains and a total of 50 anaerobic bacteria clinical isolates comprising ten different genera and 14 species were identified and analyzed by the ANC card together with Vitek 2 identification system and Vitek MS together with version 2.0 database respectively. 16S rRNA gene sequencing was used as reference method for accuracy in the identification.

Results

Vitek 2 ANC card and Vitek MS provided comparable results at species level for the five reference strains. Of 50 clinical strains, the Vitek MS provided identification for 46 strains (92%) to the species level, 47 (94%) to genus level, one (2%) low discrimination, two (4%) no identification and one (2%) misidentification. The Vitek 2 ANC card provided identification for 43 strains (86%) correct to the species level, 47 (94%) correct to the genus level, three (6%) low discrimination, three (6%) no identification and one (2%) misidentification.

Conclusions

Both Vitek MS and Vitek 2 ANC card can be used for accurate routine clinical anaerobe identification. Comparing to the Vitek 2 ANC card, Vitek MS is easier, faster and more economic for each test. The databases currently available for both systems should be updated and further developed to enhance performance.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of beta-hemolytic streptococci

Objective

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as promising technology for species identification. The purpose of this investigation was to compare the performance of MS and the traditional method for identification of beta-hemolytic streptococci (BHS).

Methods

Clinical BHS isolates were identified by the BD Phoenix SMIC/ID Streptococcal panels, and two MALDI-TOF MS platforms: the VITEK MS and the Bruker MALDI Biotyper systems respectively. In case of discordant results, 16sRNA sequencing was performed to provide the reference ID.

Results

A total of 96 isolates of BHS were analyzed. Thirty-six isolates (20.8%) were re-tested by BD Phoenix for identification failure; and four isolates (4.2%) were rerun on the Bruker system for low identification score. No isolate need a second run for identification by Vitek MS system. Overall, BD Phoenix, BioTyper and Vitek MS automated system accurately identified 76 strains (79.2%), 91 (94.7%) strains and 92 (95.8%) strains, respectively.

Conclusions

Our study suggests that MALDI-TOF MS is a superior method to conventional phenotypic methods for BHS identification.     

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.     

Performance of mass spectrometric identification of bacteria and yeasts routinely isolated in a clinical microbiology laboratory using MALDI-TOF MS

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to identifying bacterial and yeast strains. The aim of this study was to evaluate the clinical performance of the VITEK^® MS system in the identification of bacteria and yeast strains routinely isolated from clinical samples.

Methods

We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria and yeasts regardless of phylum or source of isolation. Discordant results were resolved with 16S rDNA or internal transcribed spacer (ITS) gene sequencing. Colonies (a single deposit on a MALDI disposable target without any prior extraction step) were analyzed using the VITEK^® MS system. Peptide spectra acquired by the system were compared with the VITEK^® MS IVD database Version 2.0, and the identification scores were recorded.

Results

Of the 1,181 isolates (1,061 bacterial isolates and 120 yeast isolates) analyzed, 99.5% were correctly identified by MALDI-TOF mass spectrometry; 95.7% identified to the species level, 3.6% identified to the genus level, and 0.3% identified within a range of species belonging to different genera. Conversely, 0.1% of isolates were misidentified and 0.4% were unidentified, partly because the species were not included in the database. Re-testing using a second deposit provided a successful identification for 0.5% of isolates unidentified with the first deposit. Our results show that the VITEK^® MS system has exceptional performance in identifying bacteria and yeast by comparing acquired peptide spectra to those contained in its database.

Conclusions

MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive method for bacterial and yeast identification. Our results demonstrate that the VITEK^® MS system is a fast and reliable technique, and has the potential to replace conventional phenotypic identification for most bacterial and yeast strains routinely isolated in clinical microbiology laboratories.