Investigation of the inconsistent results between two methods in determining fluoroquinolone resistance against Mycobacterium tuberculosis isolates

doi:10.19982/j.issn.1000-6621.20230161

Abstract

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

CLC Number:

R446.5

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

Figures/Tables 3

References 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

类型/ 菌株编号	左氧氟沙星		莫西沙星		熔解曲线法	全基因组测序	突变与耐药相关的可信等级^[5]
类型/ 菌株编号	最低抑菌浓度 (μg/ml)	表型药敏	最低抑菌浓度 (μg/ml)	表型药敏	熔解曲线法	(突变频率,%)^a	突变与耐药相关的可信等级^[5]
Lfx和(或)Mfx表型药敏试验耐药/熔解曲线法敏感
22A010	1	敏感	0.5	耐药	敏感	None	-
22A064	1	敏感	0.5	耐药	敏感	gyrB_Arg446Leu(99.1)	不明确相关
22A018	2	耐药	0.5	耐药	敏感	gyrB_Asp461Asn(100.0)	中等相关
22A106	2	耐药	0.5	耐药	敏感	None	-
22A214	4	耐药	1.0	耐药	敏感	gyrA_Asp94Ala(15.3)	明确相关
Lfx和(或)Mfx表型药敏试验敏感/熔解曲线法耐药
22A043	1	敏感	0.5	耐药	耐药	gyrA_Asp94Ala(100.0)	明确相关
22A109	1	敏感	0.5	耐药	耐药	gyrA_Ala90Val(99.4)	明确相关
22A036	2	耐药	0.25	敏感	耐药	gyrA_Asp94Asn(100.0)	明确相关
22A076	2	耐药	0.25	敏感	耐药	gyrA_Ala90Val(100.0)	明确相关
22A079	2	耐药	0.25	敏感	耐药	gyrA_Ala90Val(100.0)	明确相关
22A117	2	耐药	0.25	敏感	耐药	gyrA_Ala90Val(100.0)	明确相关
22A135	2	耐药	0.25	敏感	耐药	gyrA_Ala90Val(99.2)	明确相关