两种结核分枝杆菌氟喹诺酮类药物耐药性检测方法比较及不一致原因初探

doi:10.19982/j.issn.1000-6621.20230161

摘要/Abstract

摘要：

目的: 评估荧光PCR熔解曲线法(简称“熔解曲线法”)对结核分枝杆菌氟喹诺酮类药物(FQs)耐药性的诊断价值,分析其与表型药物敏感性(简称“药敏”)试验结果不一致的原因。方法: 采用简单随机抽样的方法,随机选取2019年1月至2020年6月期间来自重庆市39个区(县)经菌种鉴定和比例法药敏试验确定为耐多药结核病的126例患者的临床分离株,分别采用微量肉汤稀释法和熔解曲线法对左氧氟沙星(Lfx)和莫西沙星(Mfx)进行表型药敏及分子药敏试验。以表型药敏试验结果为标准,评价熔解曲线法对FQs耐药的检测效能,并采用全基因组测序(WGS)对表型药敏和分子药敏试验不一致的结果进行分析。结果: 以表型药敏试验结果为标准,熔解曲线法对FQs耐药检测的敏感度和特异度分别为94.5%(86/91;95%CI:87.1%~98.0%)和100.0%(35/35;95%CI:87.7%~100.0%)。两种方法结果不一致者有12株,不一致率为9.5%(12/126)。表型药敏试验耐药而熔解曲线法敏感者5株,WGS显示2株未见gyrA耐药基因相关突变,2株存在gyrB基因突变(该4株菌的最低抑菌浓度值,Lfx为1~2μg/ml,Mfx均为0.5μg/ml),1株发现gyrA基因Asp94Ala突变,突变频率为15.3%。表型药敏试验敏感而熔解曲线法耐药7株,其中5株发现gyrA基因Ala90Val突变,1株发现gyrA基因Asp94Ala突变,1株发现gyrA基因Asp94Asn突变,该7株菌对Lfx和Mfx的最低抑菌浓度范围分别为1~2μg/ml和0.25~0.5μg/ml。结论: 熔解曲线法对FQs耐药的检测效能较好,FQs异质性耐药菌的比例及低水平耐药相关突变是影响FQs熔解曲线法检测效能及导致其与表型药敏试验结果不一致的主要原因。

关键词: 分枝杆菌, 结核, 喹诺酮类, 核酸扩增技术, 结核, 抗多种药物性

Abstract:

Objective: To evaluate the performance of the fluorescence PCR melting curve method in determining fluoroquinolone resistance in Mycobacterium tuberculosis isolates, and to analyze the inconsistency between the fluorescence PCR melting curve method (melting curve method) and the phenotypic drug susceptibility test (DST) in detecting the susceptibility of fluoroquinolone. Methods: A total of 126 multidrug-resistant tuberculosis (MDR-TB) isolates confirmed with identification and DST from 39 counties of Chongqing City between January 2019 and June 2020 were randomly selected using simple random sampling. Phenotypic DST by both microdilution testing and melting curve method were applied to detect the drug resistance to levofloxacin (Lfx) and moxifloxacin (Mfx). Phenotypic DST served as a reference standard to assess the detection performance. Whole genome sequencing (WGS) was used to analyze the discordant results between the phenotypic and molecular DST. Results: Taking the phenotypic DST as the standard, the sensitivity, specificity of melting curve method were 94.5% (86/91, 95%CI: 87.1%-98.0%), 100.0% (35/35, 95%CI: 87.7%-100.0%) respectively. There were 12 cases with discordant results between the two methods, and the inconsistency rate was 9.5% (12/126). Five isolates identified as FQs-resistant by phenotypic DST showed melting curve method susceptibility. Further WGS sequencing analysis showed that 2 isolates didn’t harbor any gyrA mutations, 2 isolates harbored gyrB mutations (the MIC of these 4 isolates were 1-2 μg/ml for Lfx and 0.5 μg/ml for Mfx) and 1 isolate with 15.3% FQs heteroresistance harbored gyrA_Asp94Ala mutation. Seven isolates identified as FQs-resistant by melting curve method showed phenotypic susceptibility to FQs. WGS sequencing analysis detected 5 isolates with gyrA_Ala90Val, 1 with gyrA_Asp94Ala and 1 with gyrA_Asp94Asn, the MIC of these 7 isolates were 1-2 μg/ml for Lfx and 0.25-0.5 μg/ml for Mfx. Conclusion: The melting curve method showed high detection efficiency for FQs resistance. The proportion of FQs heteroresistant populations and the low-level-resistance-associated mutations are the main reasons that affect the detection performance of melting curve method and lead to its inconsistency with phenotypic DST.

Key words: Mycobacterium tuberculosis, Quinolones, Nucleic acid amplification techniques, Tuberculosis, multidrug-resistant

中图分类号:

R446.5

詹建, 游国庆, 何霞, 孔珏颖, 刘文果, 冯鑫, 胡彦. 两种结核分枝杆菌氟喹诺酮类药物耐药性检测方法比较及不一致原因初探[J]. 中国防痨杂志, 2023, 45(11): 1058-1063. doi: 10.19982/j.issn.1000-6621.20230161

Zhan Jian, You Guoqing, He Xia, Kong Jueying, Liu Wenguo, Feng Xin, Hu Yan. Investigation of the inconsistent results between two methods in determining fluoroquinolone resistance against Mycobacterium tuberculosis isolates[J]. Chinese Journal of Antituberculosis, 2023, 45(11): 1058-1063. doi: 10.19982/j.issn.1000-6621.20230161

图/表 3

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参考文献 17

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熔解曲线法	表型药敏试验		合计 (株)	敏感度 (%,95%CI)	特异度 (%,95%CI)	阳性预测值 (%,95%CI)	阴性预测值 (%,95%CI)	一致率 (%,95%CI)	Kappa值
熔解曲线法	耐药(株)	敏感(株)	合计 (株)	敏感度 (%,95%CI)	特异度 (%,95%CI)	阳性预测值 (%,95%CI)	阴性预测值 (%,95%CI)	一致率 (%,95%CI)	Kappa值
Lfx				96.6 (89.6~99.1)	94.9 (81.4~99.1)	97.7 (91.1~99.6)	92.5 (78.5~98.0)	96.0 (90.5~98.5)	0.908
耐药	84	2	86
敏感	3	37	40
合计	87	39	126
Mfx				94.2 (86.3~97.8)	87.5 (72.4~95.3)	94.2 (86.3~97.8)	87.5 (72.4~95.3)	92.1 (85.5~96.0)	0.817
耐药	81	5	86
敏感	5	35	40
合计	86	40	126
FQs				94.5 (87.1~98.0)	100.0 (87.7~100.0)	100.0 (94.7~100.0)	87.5 (72.4~95.3)	96.0 (90.5~98.5)	0.905
耐药^a	86	0	86
敏感^b	5	35	40
合计	91	35	126