Application of matrix assisted laser desorption ionization time of flight mass spectrometry in the identification of <i>Mycobacterium tuberculosis</i>_West China Medical Journal

Authors：

DAI Zhongqiu , TANG Sishi , FAN Yujie , SONG Qifei , WANG Yuanfang , YUAN Yu , SHU Ling ,  XIE Yi

Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

XIE Yi, Email: xie_yi_77@163.com

Keywords：

Matrix assisted laser desorption ionization time of flight mass spectrometry; Identification of mycobacteria; Mycobacterium tuberculosis complex; Non-tuberculous mycobacteria

DOI：

10.7507/1002-0179.201907080

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Objective To evaluate the accuracy and practicability of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) in clinical isolates of mycobacteria.Methods We collected all tested strains, which were positive for Mycobacterium tuberculosis culture and positive for acid-fast staining, from West China Hospital of Sichuan University from 2014 to 2017, eliminating duplicate strains sent by the same patient at the same time. The traditional method was used with the P-nitrobenzoic acid (PNB)/ 2-Thiophenecarboxylic acid hydrazide (TCH) indicator to initially identify acid-resistant positive strains. Mycobacteria was identified by MALDI-TOF MS; the specificity and sensitivity of the MALDI-TOF MS was analyzed by duplex primer-polymerase chain reaction (Duplex-PCR) method and DNA sequencing method as the "gold standard" for the identification.Results A total of 237 anti-acid positive strains were collected; Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacteria (NTM) were identified by mycobacterium double primer PCR, and NTM was identified by 16S rRNA gene sequencing. There were 218 cases of MTC and 19 cases of NTM. The results of preliminary identification using the traditional identification method of PNB/TCH indicator showed that there were 209 cases of MTC (with the sensitivity of 95.9%, specificity of 100.0%, positive predictive value of 100.0%, and negative predictive value of 67.9%) and 28 cases of NTM (with the sensitivity of 100.0%, specificity of 95.9%, positive predictive value of 67.9%, and negative predictive value of 100.0%). The results of MALDI-TOF MS method indicated that there were 199 cases of MTC (with the sensitivity of 91.3%, specificity of 100.0%, positive predictive value of 100.0%, and negative predictive value of 50.0%), 32 cases of NTM (with the sensitivity of 68.4%. specificity of 94.0%, positive predictive value of 40.6%, and negative predictive value of 97.1%), and 6 cases of others. There were 168 strains (84.4%) with the identification score>1.9 obtained by MALDI-TOF MS method.Conclusions MALDI-TOF MS is a better method for identifying mycobacteria, which has the same identification results as the traditional methods, and has low cost and is suitable for routine use in clinical microbiology laboratories.

Citation： DAI Zhongqiu, TANG Sishi, FAN Yujie, SONG Qifei, WANG Yuanfang, YUAN Yu, SHU Ling, XIE Yi. Application of matrix assisted laser desorption ionization time of flight mass spectrometry in the identification of Mycobacterium tuberculosis. West China Medical Journal, 2019, 34(8): 844-849. doi: 10.7507/1002-0179.201907080 Copy

1.	World Health Organization. Global tuberculosis report 2017. (2017-10-30)[2019-07-03]. https://www.who.int/tb/publications/global_report/gtbr2017_main_text.pdf.
2.	全国第五次结核病流行病学抽样调查技术指导组, 全国第五次结核病流行病学抽样调查办公室. 2010年全国第五次结核病流行病学抽样调查报告. 中国防痨杂志, 2012, 34(8): 485-508.
3.	李玉静, 马京华, 张彦坤, 等. 8种结核病实验室检测方法诊断价值的对比分析. 医学动物防制, 2017, 33(1): 25-28, 32.
4.	Body BA, Beard MA, Slechta ES, et al. Evaluation of the vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of mycobacterium and nocardia species. J Clin Microbiol, 2018, 56(6): pii: e00237-18.
5.	中国防痨协会. 结核病诊断细菌学检验规程. 中国防痨杂志, 1996, 18(2): 80-85.
6.	李道群, 夏雪山, 宋玉竹, 等. 结核分枝杆菌分子分型技术的研究和应用. 中国人兽共患病学报, 2016, 32(1): 76-82.
7.	Kim BJ, Hong SK, Lee KH, et al. Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB). J Clin Microbiol, 2004, 42(3): 1308-1312.
8.	Lee AS, Jelfs P, Sintchenko V, et al. Identification of non-tuberculous mycobacteria: utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRNA gene sequencing. J Med Microbiol, 2009, 58(Pt 7): 900-904.
9.	Petti CA, Bosshard PP, Brandt ME, et al. Interpretive criteria for identification of bacteria and fungi by DNA target sequencing; approved guideline. Clinical and Laboratory Standards Institute (CLSI), 2008.
10.	Keys CJ, Dare DJ, Sutton H, et al. Compilation of a MALDI-TOF Mass spectral database for the rapid screening and characterization of bacteria implicated in human infectious diseases. Infect Genet Evol, 2004, 4(3): 221-224.
11.	Mediavilla-Gradolph MC, De Toro-Peinado I, Bermúdez-Ruiz MP, et al. Use of MALDI-TOF MS for identification of nontuberculous mycobacterium species isolated from clinical specimens. BioMed Res Int, 2015, 2015: 854078.
12.	周昭彦, 胡必杰, 鲍容, 等. MALDI-TOF质谱快速鉴定非结核分枝杆菌方法的建立和评价. 中华检验医学杂志, 2013, 36(7): 610-614.
13.	Huang TS, Lee CC, Tu HZ, et al. Rapid identification of mycobacteria from positive MGIT broths of primary cultures by MALDI-TOF mass spectrometry. PLoS ONE, 2018, 13(2): e0192291.
14.	刘国威, 白大鹏, 梅早仙. 非结核分枝杆菌肺病的诊断与治疗研究进展. 中国城乡企业卫生, 2016, 31(6): 18-21.
15.	刘国标, 罗春明, 邓虹, 等. 抗结核治疗对非结核分枝杆菌肺病的疗效观察. 现代医院, 2017, 17(3): 354-356.
16.	Vijay GS Kumar, Tejashree A Urs, Rajani R Ranganath. MPT 64 antigen detection for rapid confirmation of M.tuberculosis isolates. BMC Res Notes, 2011, Vol.4(1).
17.	Şamlı A, İlki A. Comparison of MALDI-TOF MS, nucleic acid hybridization and the MPT64 immunochromatographic test for the identificationof M. Tuberculosis and non-tuberculosis Mycobacterium species. New Microbiol, 2016, 4(39): 259-263.
18.	王雷, 施旭东. 水溶性对硝基苯甲酸在结核分枝杆菌菌种鉴定中的应用. 临床医学工程, 2010, 17(11): 47-48.
19.	蔡静, 张鑫, 魏黛玉, 等. 甘肃省53 株非结核分枝杆菌对9 种常用抗结核药物的敏感性分析. 疾病监测, 2018, 33(6): 515-519.
20.	Carbonnelle E, Mesquita C, Bille E, et al. MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory. Clin Biochem, 2010, 44(1): 104-109.
21.	Akyar I, Çavuşoğlu C, Ayaş M, et al. Evaluation of the performance of MALDI-TOF MS and DNA sequence analysis in the identification of mycobacteria species. Turk J Med Sci, 2018, 48(6): 1351-1357.
22.	van Veen SQ, Claas EC, Kuijper EJ, et al. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol, 2010, 48(3): 900-907.
23.	王胜芬, 赵雁林, 黄海荣, 等. 结核分枝杆菌北京基因型菌株与耐药表型的关系. 中国医学科学院学报, 2009, 31(4): 427-431.
24.	刘栋宾, 向延根, 马小华, 等. 结核分枝杆菌L型特征及临床意义研究进展. 实用预防医学, 2018, 25(9): 1150-1153, 封3.
25.	Genc GE, Demir M, Yaman G, et al. Evaluation of MALDI-TOF MS for identification of nontuberculous mycobacteria isolated from clinical specimens in mycobacteria growth indicator tube medium. New Microbiol, 2018, 41(3): 214-219.

1. World Health Organization. Global tuberculosis report 2017. (2017-10-30)[2019-07-03]. https://www.who.int/tb/publications/global_report/gtbr2017_main_text.pdf.
2. 全国第五次结核病流行病学抽样调查技术指导组, 全国第五次结核病流行病学抽样调查办公室. 2010年全国第五次结核病流行病学抽样调查报告. 中国防痨杂志, 2012, 34(8): 485-508.
3. 李玉静, 马京华, 张彦坤, 等. 8种结核病实验室检测方法诊断价值的对比分析. 医学动物防制, 2017, 33(1): 25-28, 32.
4. Body BA, Beard MA, Slechta ES, et al. Evaluation of the vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of mycobacterium and nocardia species. J Clin Microbiol, 2018, 56(6): pii: e00237-18.
5. 中国防痨协会. 结核病诊断细菌学检验规程. 中国防痨杂志, 1996, 18(2): 80-85.
6. 李道群, 夏雪山, 宋玉竹, 等. 结核分枝杆菌分子分型技术的研究和应用. 中国人兽共患病学报, 2016, 32(1): 76-82.
7. Kim BJ, Hong SK, Lee KH, et al. Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB). J Clin Microbiol, 2004, 42(3): 1308-1312.
8. Lee AS, Jelfs P, Sintchenko V, et al. Identification of non-tuberculous mycobacteria: utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRNA gene sequencing. J Med Microbiol, 2009, 58(Pt 7): 900-904.
9. Petti CA, Bosshard PP, Brandt ME, et al. Interpretive criteria for identification of bacteria and fungi by DNA target sequencing; approved guideline. Clinical and Laboratory Standards Institute (CLSI), 2008.
10. Keys CJ, Dare DJ, Sutton H, et al. Compilation of a MALDI-TOF Mass spectral database for the rapid screening and characterization of bacteria implicated in human infectious diseases. Infect Genet Evol, 2004, 4(3): 221-224.
11. Mediavilla-Gradolph MC, De Toro-Peinado I, Bermúdez-Ruiz MP, et al. Use of MALDI-TOF MS for identification of nontuberculous mycobacterium species isolated from clinical specimens. BioMed Res Int, 2015, 2015: 854078.
12. 周昭彦, 胡必杰, 鲍容, 等. MALDI-TOF质谱快速鉴定非结核分枝杆菌方法的建立和评价. 中华检验医学杂志, 2013, 36(7): 610-614.
13. Huang TS, Lee CC, Tu HZ, et al. Rapid identification of mycobacteria from positive MGIT broths of primary cultures by MALDI-TOF mass spectrometry. PLoS ONE, 2018, 13(2): e0192291.
14. 刘国威, 白大鹏, 梅早仙. 非结核分枝杆菌肺病的诊断与治疗研究进展. 中国城乡企业卫生, 2016, 31(6): 18-21.
15. 刘国标, 罗春明, 邓虹, 等. 抗结核治疗对非结核分枝杆菌肺病的疗效观察. 现代医院, 2017, 17(3): 354-356.
16. Vijay GS Kumar, Tejashree A Urs, Rajani R Ranganath. MPT 64 antigen detection for rapid confirmation of M.tuberculosis isolates. BMC Res Notes, 2011, Vol.4(1).
17. Şamlı A, İlki A. Comparison of MALDI-TOF MS, nucleic acid hybridization and the MPT64 immunochromatographic test for the identificationof M. Tuberculosis and non-tuberculosis Mycobacterium species. New Microbiol, 2016, 4(39): 259-263.
18. 王雷, 施旭东. 水溶性对硝基苯甲酸在结核分枝杆菌菌种鉴定中的应用. 临床医学工程, 2010, 17(11): 47-48.
19. 蔡静, 张鑫, 魏黛玉, 等. 甘肃省53 株非结核分枝杆菌对9 种常用抗结核药物的敏感性分析. 疾病监测, 2018, 33(6): 515-519.
20. Carbonnelle E, Mesquita C, Bille E, et al. MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory. Clin Biochem, 2010, 44(1): 104-109.
21. Akyar I, Çavuşoğlu C, Ayaş M, et al. Evaluation of the performance of MALDI-TOF MS and DNA sequence analysis in the identification of mycobacteria species. Turk J Med Sci, 2018, 48(6): 1351-1357.
22. van Veen SQ, Claas EC, Kuijper EJ, et al. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol, 2010, 48(3): 900-907.
23. 王胜芬, 赵雁林, 黄海荣, 等. 结核分枝杆菌北京基因型菌株与耐药表型的关系. 中国医学科学院学报, 2009, 31(4): 427-431.
24. 刘栋宾, 向延根, 马小华, 等. 结核分枝杆菌L型特征及临床意义研究进展. 实用预防医学, 2018, 25(9): 1150-1153, 封3.
25. Genc GE, Demir M, Yaman G, et al. Evaluation of MALDI-TOF MS for identification of nontuberculous mycobacteria isolated from clinical specimens in mycobacteria growth indicator tube medium. New Microbiol, 2018, 41(3): 214-219.

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