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

doi:10.19982/j.issn.1000-6621.20230113

摘要/Abstract

摘要：

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。

关键词: 核酸类, 质谱分析法, 结核, 分枝杆菌感染, 总结性报告(主题)

Abstract:

Nucleic acid matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique has been increasingly applied in the etiology and drug resistance diagnosis of tuberculosis and non-tuberculous mycobacteriosis, providing a rapid and accurate basis for early diagnosis, differential diagnosis, and drug resistance identification. However, it needs to be normalized that the clinician’s understanding and grasp of sample selection, retention, submitting time and precautions, and interpretation of report results. This consensus summarizes the clinical indications and specimen collection precautions of nucleic acid MALDI-TOF MS detection technique for the diagnosis of tuberculosis and non-tuberculous mycobacteriosis, and introduces how to correctly interpret the reported results of nucleic acid MALDI-TOF MS technique used to mycobacterial species and drug resistance. It has important significance to further standardize the clinical application of nucleic acid MALDI-TOF MS technique in the diagnosis of tuberculosis and non-tuberculous mycobacteriosis, improve the level of clinical diagnosis, and guide the early accurate and effective clinical treatment.

Key words: Nucleic acids, Mass spectrometry, Tuberculosis, Mycobacterium infections, Consensus development conferences as topic

中图分类号:

中国人民解放军总医院第八医学中心结核病医学部, 《中国防痨杂志》编辑委员会, 中国医疗保健国际交流促进会结核病防治分会. 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[J]. 中国防痨杂志, 2023, 45(6): 543-558. doi: 10.19982/j.issn.1000-6621.20230113

Senior Department of Tuberculosis, the th Medical Center of Chinese PLA General Hospital, Editorial Board of Chinese Journal of Antituberculosis, Tuberculosis Control Branch of China Intrnational Exchange and Promotive Association for Medical and Health Care. Expert consensus on the clinical application of nucleic acid MALDI-TOF MS technique in the diagnosis of tuberculosis and non-tuberculosis mycobacteriosis[J]. Chinese Journal of Antituberculosis, 2023, 45(6): 543-558. doi: 10.19982/j.issn.1000-6621.20230113

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诊断方法	应用优势	应用局限
实时荧光定量PCR技术	可直接检测呼吸道样本,技术成熟,应用广泛,敏感度和特异度均较高	检测通量较低
恒温扩增技术	无需温度梯度循环,保持温度恒定,对设备要求不高。快速检测结核分枝杆菌,肉眼可观察	仅能进行病原学的鉴定,不能检测耐药性。高温、高湿、试剂不足和交叉污染会出现假阳性结果
基因芯片/线性探针技术	高通量,可一次完成菌种鉴定和多个耐药相关基因位点的检测。敏感度和特异度均较高	所需样本量大。检测的耐药位点有限;杂交、洗涤过程较繁琐、费时
基因测序技术	宏基因组测序可同时检测多种病原微生物,可检测到已知耐药位点以外的突变,可用于发现新的耐药机制。靶向测序技术可检测到所有的耐药相关基因的突变类型。一代测序常作为分子药物敏感性检测结果和表型药物敏感性检测结果不一致时的验证手段	靶向测序技术对样本、设备和结果解读的要求高。一代测序对异质性耐药的检测受限
核酸MALDI-TOF MS技术	高通量,可一次性覆盖所有一线药物和大部分常用二线药物的耐药基因位点,可很好区分结核分枝杆菌和非结核分枝杆菌,具有较高的灵活性。敏感度和特异度高。可检测混合感染、异质性耐药、同义突变	只能覆盖已知的耐药基因位点,依赖于耐药机制的发现

序号	中文名称	拉丁文名称
结核分枝杆菌复合群
1	结核分枝杆菌	M.tuberculosis
2	牛分枝杆菌	M.bovis
3	牛分枝杆菌BCG	M.bovis BCG
4	田鼠分枝杆菌	M.microti
5	非洲分枝杆菌	M.africanum
6	卡内蒂分枝杆菌	M.canetti
7	山羊分枝杆菌	M.caprae
8	海豹分枝杆菌	M.pinnipedii
非结核分枝杆菌
1	鸟分枝杆菌	M.avium
2	胞内分枝杆菌	M.intracellulare
3	莲建洞分枝杆菌	M.yongonense
4	脓肿分枝杆菌	M.abscessus
5	博莱分枝杆菌	M.bolletii
6	马赛分枝杆菌	M.massiliense
7	偶发分枝杆菌	M.fortuitum
8	堪萨斯分枝杆菌	M.kansasii
9	戈登分枝杆菌	M.gordonae
10	瘰疬分枝杆菌	M.scrofulaceum
11	龟分枝杆菌	M.chelonae
12	海分枝杆菌	M.marinum
13	蟾蜍分枝杆菌	M.xenopi
14	溃疡分枝杆菌	M.ulcerans
15	嗜血分枝杆菌	M.haemophilum
16	玛尔摩分枝杆菌	M.malmoense
17	耻垢分枝杆菌	M.smegmatis
18	苏尔加分枝杆菌	M.szulgai
19	不产色分枝杆菌	M.nonchromogenicum
20	次要分枝杆菌	M.triviale
21	土分枝杆菌	M.terrae
22	猪分枝杆菌	M.porcinum
23	浅黄分枝杆菌	M.gilvum
24	草分枝杆菌	M.phlei
25	胃分枝杆菌	M.gastri
26	母牛分枝杆菌	M.vaccae
27	迪氏分枝杆菌	M.diernhoferi
28	猿分枝杆菌	M.simiae
29	新金色分枝杆菌	M.neoaurum
30	金色分枝杆菌	M.aurum
31	居间分枝杆菌	M.interjectum
32	奇美拉分枝杆菌	M.chimaera
33	免疫原分枝杆菌	M.immunogenum
34	产黏液分枝杆菌	M.mucogenicum
35	外来分枝杆菌	M.peregrinum
36	三重分枝杆菌	M.triplex
37	布兰德分枝杆菌	M.branderi
38	慢生黄分枝杆菌	M.lentiflavum
39	副瘰疬分枝杆菌	M.parascrofulaceum
40	中间分枝杆菌	M.intermedium

药物名称	耐药基因^a	检测位点	密码子/碱基	编码氨基酸
利福平	rpoB^a	511	CTG	亮氨酸(Leu)
		513	CAA	谷氨酰胺(Gln)
		516	GAC	天冬氨酸(Asp)
		522	TCG	丝氨酸(Ser)
		526	CAC	组氨酸(His)
		531	TCG	丝氨酸(Ser)
		533	CTG	亮氨酸(Leu)
		572	ATC	异亮氨酸(Ile)
异烟肼	inhA	-15	C	/
	katG	315	AGC	丝氨酸(Ser)
		316	GGC	甘氨酸(Gly)
吡嗪酰胺	pncA	57	CAC	组氨酸(His)
		-11	A	/
乙胺丁醇	embB	306	ATG	蛋氨酸(Met)
		406	GGC	甘氨酸(Gly)
氟喹诺酮类	gyrA	88	GGC	甘氨酸(Gly)
		90	GCG	丙氨酸(Ala)
		91	TCG	丝氨酸(Ser)
		94	GAC	天冬氨酸(Asp)
	gyrB	538	AAC	天冬酰胺(Asn)
链霉素	rpsL	43	AAG	赖氨酸(Lys)
		88	AAG	赖氨酸(Lys)
乙硫异烟胺/丙硫异烟胺	inhA	-15	C	/
对氨基水杨酸钠	folC	43	ATC	异亮氨酸(Ile)
	thyA	202	ACC	苏氨酸(Thr)
		75	CAC	组氨酸(His)
阿米卡星	rrs	1401	A	/
		1484	G	/
卡那霉素	eis	-14	C	/
	rrs	1401	A	/
		1402	C	/
		1484	G	/
卷曲霉素	rrs	1401	A	/
		1402	C	/
		1484	G	/
环丝氨酸	alr	261	AGC	丝氨酸(Ser)
		113	CTG	亮氨酸(Leu)
	ald	32	GAA	谷氨酸(Glu)
氯法齐明	rv0678	193	G	/
		466	C	/
贝达喹啉	rv0678	193	G	/
		466	C	/
利奈唑胺	rplC	460	T	/

药物名称	耐药基因^a	检测位点	密码子/碱基	编码氨基酸
氨基糖苷类(卡那霉素、阿米卡星、庆大霉素)	rrs^a	1406	T	/
		1408	A	/
		1491	G	/
大环内酯类(阿奇霉素、克拉霉素)	erm(41)	28	T	/
	rrl^a	2058	A	/
		2059	A	/

耐药基因	检测位点	野生型密码子 (氨基酸)	突变型密码子 (氨基酸)
rpoB	513	CAA(谷氨酰胺)	CAG(谷氨酰胺)
	533	CTG(亮氨酸)	TTG(亮氨酸)
gyrA	90	GCG(丙氨酸)	GCA(丙氨酸)
	94	GAC(天冬氨酸)	GAT(天冬氨酸)