多色探针熔解曲线分析技术检测准广泛耐药肺结核的应用价值

doi:10.19982/j.issn.1000-6621.20220418

摘要/Abstract

摘要：

目的: 评价多色探针熔解曲线分析技术(multicolor melting curve analysis,MMCA)在准广泛耐药肺结核(pre-extensive drug-resistant pulmonary tuberculosis,pre-XDR-PTB)检测中的应用价值。方法: 搜集2019年1月至2020年12月深圳市慢性病防治中心及深圳市各区级结核病防治机构登记的培养阳性且初次被鉴定为耐多药/利福平耐药肺结核(multidrug-/rifampicin-resistant pulmonary tuberculosis,MDR/RR-PTB)的129例患者的129份痰标本。对标本进行3种氟喹诺酮类(fluoroquinolones,FQs)药物[氧氟沙星(ofloxacin,Ofx)、左氧氟沙星(levofloxacin,Lfx)、莫西沙星(moxifloxacin,Mfx)]的表型药物敏感性试验(简称“表型药敏试验”)和MMCA检测,并使用Sanger测序方法对标本的检测结果进行验证。以表型药敏试验结果为参照,分析MMCA检测MDR/RR-PTB患者对FQs耐药性的效能。结果: 以表型药敏试验结果为参照,MMCA检测MDR/RR-PTB患者标本对Ofx耐药的敏感度为87.0%(40/46),特异度为94.0%(78/83),Kappa值为0.813;检测MDR/RR-PTB患者标本对Lfx耐药的敏感度为97.3%(36/37),特异度为90.2%(83/92),Kappa值为0.822;检测MDR/RR-PTB患者标本对Mfx耐药的敏感度为92.3%(24/26),特异度为79.6%(82/103),Kappa值为0.565。将3种FQs的表型药敏试验结果合并,任意一种耐药即判定为FQs耐药,则MMCA检测MDR/RR-PTB患者标本对FQs耐药的敏感度为85.1%(40/47),特异度为93.9%(77/82),Kappa值为0.797。对疑似耐药的52份标本进行Sanger测序,其中96.2%(50/52)的标本的测序结果与MMCA一致,3.8%(2/52)的标本在MMCA的非检测区域存在突变。结论: MMCA与FQs表型药敏试验结果的一致性较高,可以用于检测pre-XDR-PTB。

关键词: 分枝杆菌,结核, 抗药性,多种,细菌, 微生物敏感性试验, 分子诊断技术, 诊断,鉴别

Abstract:

Objective: To evaluate the application value of multicolor melting curve analysis (MMCA) in detection of pre-extensive drug-resistant pulmonary tuberculosis (pre-XDR-PTB). Methods: A total of 129 sputum samples from 129 patients with positive culture and initially identified as multidrug-/rifampicin-resistant pulmonary tuberculosis (MDR/RR-PTB) were collected. All the patients were registered in the Shenzhen Center for Chronic Disease Control and district-level tuberculosis prevention and control institutions from January 2019 to December 2020. The samples were detected by drug susceptibility tests (DST) of three fluoroquinolones (ofloxacin (Ofx), levofloxacin (Lfx), and moxifloxacin (Mfx)) and MMCA. Sanger sequencing was performed as the third-party verification of the results. Using DST as the gold standard, the efficacy of MMCA in detecting fluoroquinolone resistance in patients with MDR/RR-PTB was analyzed. Results: Using DST as the gold standard, the sensitivity, specificity, and Kappa values of the MMCA method for detecting fluoroquinolone resistance in MDR/RR-PTB patients’ samples were 87.0% (40/46), 94.0% (78/83), and 0.813, respectively, for Ofx; 97.3% (36/37), 90.2% (83/92), and 0.822, respectively, for Lfx; 92.3% (24/26), 79.6% (82/103), and 0.565, respectively, for Mfx. If combining the DST results of three fluoroquinolones and defining fluoroquinolone resistance as resistance to any one of the three fluoroquinolones, the sensitivity, specificity, and Kappa value of the MMCA method for detecting fluoroquinolone resistance in MDR/RR-PTB patients’ samples were 85.1% (40/47), 93.9% (77/82), and 0.797, respectively. Sanger sequencing was performed on 52 suspected of drug resistance samples. Among these samples, 96.2% (50/52) had the same sequencing results as MMCA, and 3.8% (2/52) of the samples had mutations in the non-detection region of MMCA. Conclusion: The results of MMCA were consistent with those of DST, and thus MMCA could be used to detect pre-XDR-PTB.

Key words: Mycobacterium tuberculosis, Drug resistance, multiple, bacterial, Microbial sensitivity tests, Molecular diagnostic techniques, Diagnosis, differential

中图分类号:

R52
R825.2

朱玉梅, 夏子涵, 李金莉, 何慧珊, 王峰. 多色探针熔解曲线分析技术检测准广泛耐药肺结核的应用价值[J]. 中国防痨杂志, 2023, 45(4): 406-411. doi: 10.19982/j.issn.1000-6621.20220418

Zhu Yumei, Xia Zihan, Li Jinli, He Huishan, Wang Feng. Application value of multicolor melting curve analysis in detection of pre-extensive drug-resistant pulmonary tuberculosis[J]. Chinese Journal of Antituberculosis, 2023, 45(4): 406-411. doi: 10.19982/j.issn.1000-6621.20220418

图/表 2

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MMCA检测	表型药敏试验				Sanger测序	标本份数
MMCA检测	FQs	Ofx	Lfx	Mfx	Sanger测序	标本份数
突变型	R	R	R	R	gyrA D94G	12
	R	R	R	R	gyrA D94N	4
	R	R	R	R	gyrA D94H	2
	R	R	R	R	gyrA S91P	2
	R	R	R	R	gyrA G88C	2
	R	R	R	R	gyrA D94Y	1
	R	R	R	R	gyrA A90V	1
	R	R	R	S	gyrA A90V	6
	R	R	R	S	gyrA D94A	2
	R	R	R	S	gyrA D94G	2
	R	R	R	S	gyrA D94N	1
	R	R	R	S	gyrA D94H	1
	R	R	S	S	gyrA D94A	3
	R	R	S	S	gyrA A90V	1
	S	S	S	S	gyrA D94A	3
	S	S	S	S	gyrA A90V	2
野生型	R	R	R	R	gyrB E540D	1
	R	R	S	S	gyrB野生型	5
	R	S	S	R	gyrB E540D	1
	S	S	S	S	未测序(表型药敏试验结果与MMCA检测结果一致)	77