Analysis on characteristic of drug resistance-associated gene mutations and the correlation with genotypes among Mycobacterium tuberculosis isolates in Zhejiang Province

doi:10.19982/j.issn.1000-6621.20220224

Abstract

Abstract:

Objective: To evaluate the drug resistance analysis and detection performance of whole-genome sequencing (WGS) on the characteristic of drug resistance-associated gene mutations and its correlation with genotypes among Mycobacterium tuberculosis (MTB) isolates in Zhejiang Province. Methods: WGS analysis of 14 drugs was performed on 808 MTB strains collected from the fifth tuberculosis drug resistance surveillance project in Zhejiang Province from 2018 and 2019. MTB genotypes and drug resistance-related gene mutations were identified, meanwhile, their relationship and the consistency of WGS drug susceptibility test results and phenotypic drug susceptibility test results of isoniazid were analyzed. Results: Of the 808 MTB strains, 153 were identified with drug-resistant gene mutations related to 11 anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol, pyrazinamide, streptomycin, fluoroquinolones, amikacin, kanamycin, capreomycin, ethionamide, para-aminosalicylic acid) and the overall mutation rate was 18.9%. The major drug resistance-associated gene mutation types were katG-315-S/T (isoniazid, 60.0% (45/75)), rpoB-450-S/L (rifampicin, 57.1% (16/28)), embB-306-M/V (ethambutol, 43.8% (7/16)), rpsL-43-K/R (streptomycin, 65.5% (36/55)), gyrA-94-D/G (fluoroquinolones, 36.6% (15/41)), rrs-1402-C/A (amikacin, 3/3), rrs-1402-C/A (kanamycin, 3/4), rrs-1402-C/A (capreomycin, 3/3), inhA-15-C/T (ethionamide, 65.0% (13/20)) and thyA-75-H/N (para-aminosalicylic acid, 7/8). Drug resistance-associated mutations on rpsL gene were detected only in Beijing genotype strains (6.8% (40/586) vs. 0.0% (0/222), χ²=15.943, P=0.000). The mutation rate of katG-315 was higher in Beijing genotype strains (6.8% (40/586)) than non-Beijing genotype strains (3.2% (7/222)) and the difference was statistically significant (χ²=3.964, P=0.046). The sensitivity, specificity, positive predictive value, negative predictive value, consistency rate and Kappa value of the WGS drug resistance profiling method for isoniazid were 87.5% (49/56), 98.0% (680/694), 77.8% (49/63), 99.0% (680/687), 97.2% (729/750) and 0.808, respectively. Conclusion: The major drug resistance-associated gene mutations of 11 drugs were on katG, rpoB, embB, pncA, rpsL, gyrA, rrs, thyA and inhA among Zhejiang Province MTB strains. Mutations on rpsL, katG-315 and rpsL-43 were related with Beijing genotypes. The WGS drug resistance profiling method has comprehensive detection performance for isoniazid, but the performance of some drug-resistant gene mutations is poor.

Key words: Mycobacterium tuberculosis, Drug resistance,bacterial, Sequence analysis,DNA, Mutation, Genotype

CLC Number:

Wu Kunyang, Lu Yewei, Zhang Mingwu, Zhu Yelei, Li Xiangchen, Pan Junhang, Wang Xiaomeng, Wang Wei, Jiang Minmin, Peng Xiaojun, Wang Weixin, Gao Junshun, Liu Zhengwei. Analysis on characteristic of drug resistance-associated gene mutations and the correlation with genotypes among Mycobacterium tuberculosis isolates in Zhejiang Province[J]. Chinese Journal of Antituberculosis, 2022, 44(11): 1126-1134. doi: 10.19982/j.issn.1000-6621.20220224

Figures/Tables 6

References 27

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药品	耐药菌株株数	耐药率(%)	耐药基因	突变菌株数	突变率(%)
异烟肼	75	9.3	katG	54	72.0
			inhA	14	18.7
			inhA+katG	2	2.7
			ahpC+katG	2	2.7
			ahpC	2	2.7
			ahpC+inhA+katG	1	1.3
利福平	28	3.5	rpoB	26	92.9
			rpoB+rpoC	2	7.1
乙胺丁醇	16	2.0	embB	14	87.5
			embA+embB	1	6.3
			embA	1	6.3
吡嗪酰胺	12	1.5	pncA	12	100.0
链霉素	55	6.8	rpsL	40	72.7
			rrs	12	21.8
			gid	2	3.6
			gid+rrs	1	1.8
氟喹诺酮类	41	5.1	gyrA	36	87.8
			gyrB	4	9.8
			gyrA+gyrB	1	2.4
阿米卡星	3	0.4	rrs	3	100.0
卡那霉素	4	0.5	rrs	3	75.0
			eis	1	25.0
卷曲霉素	3	0.4	rrs	3	100.0
药品	耐药菌株株数	耐药率(%)	耐药基因	突变菌株数	突变率(%)
乙硫异烟胺	20	2.5	inhA	16	80.0
			ethA	3	15.0
			ethA+inhA	1	5.0
对氨基水杨酸	8	1.0	thyA	7	87.5
			folC	1	12.5

药品	耐药菌株数	耐药率 (%)	突变类型	突变菌株数	突变率 (%)	平均测序深度	平均突变频率 (%)
异烟肼	75	9.3	katG-315-S/T	45	60.0	457	99.3
			inhA-15-C/T	13	17.3	423	94.5
			ahpC启动子-48-G/A	4	5.3	572	96.8
			其他	20	26.6	375	95.5
利福平	28	3.5	rpoB-450-S/L	16	57.1	385	97.7
			rpoB-452-L/P	4	14.3	393	82.5
			rpoB-430-L/P	4	14.3	329	100.0
			其他	9	32.1	470	99.9
乙胺丁醇	16	2.0	embB-306-M/V	7	43.8	396	95.5
			embB-306-M/I	2	12.5	381	100.0
			embB-354-D/A	2	12.5	491	99.9
			其他	6	37.5	483	90.6
吡嗪酰胺	12	1.5	-	-	-	526	99.6
链霉素	55	6.8	rpsL-43-K/R	36	65.5	569	99.8
			rrs-514-A/C	6	10.9	418	97.1
			rpsL-88-K/R	3	5.5	473	100.0
			其他	8	14.6	329	64.0
氟喹诺酮类	41	5.1	gyrA-94-D/G	15	36.6	473	92.9
			gyrA-90-A/V	12	29.3	491	94.5
			gyrA-94-D/N	7	17.1	572	98.7
			其他	10	24.4	495	65.4
阿米卡星	3	0.4	rrs-1402-C/A	3	3/3	249	21.7
			rrs-1484-G/T	2	2/3	290	19.3
卡那霉素	4	0.5	rrs-1402-C/A	3	3/4	249	21.7
			rrs-1484-G/T	2	2/4	290	19.3
			eis启动子-10-G/A	1	1/4	547	100.0
卷曲霉素	3	0.4	rrs-1402-C/A	3	3/3	249	21.7
			rrs-1484-G/T	2	2/3	290	19.3
乙硫异烟胺	20	2.5	inhA-15-C/T	13	65.0	423	94.5
			其他	8	40.0	469	96.6
对氨基水杨酸	8	1.0	thyA-75-H/N	7	7/8	736	99.8
			folC-150-S/G	1	1/8	613	97.4

耐药基因	北京基因型 (586株)	非北京基因型 (222株)	χ²值	P值
katG	46(7.8)	13(5.9)	0.946	0.331
rpsL	40(6.8)	0(0.0)	15.943	0.000
gyrA	26(4.4)	11(5.0)	0.099	0.753
rpoB	24(4.1)	4(1.8)	2.532	0.133

突变位点	北京基因型 (586株)	非北京基因型 (222株)	χ²值	P值
katG-315	40(6.8)	7(3.2)	3.964	0.046
rpsL-43	36(6.1)	0(0.0)	14.274	0.000
gyrA-94	18(3.1)	5(2.3)	0.391	0.532
rpoB-450	15(2.6)	1(0.5)	3.691	0.085

检测方法	表型药敏试验(株)		合计 (株)	敏感度 (%)	特异度 (%)	阳性预测值 (%)	阴性预测值 (%)	一致率 (%)	Kappa值
检测方法	耐药	敏感	合计 (株)	敏感度 (%)	特异度 (%)	阳性预测值 (%)	阴性预测值 (%)	一致率 (%)	Kappa值
全基因组测序				87.5	98.0	77.8	99.0	97.2	0.808
耐药	49	14	63
敏感	7	680	687
合计	56	694	750