儿童抗结核药物耐药比例法、微孔板法、全基因组测序检测对比研究

doi:10.12372/jcp.2023.22e0083

摘要/Abstract

摘要：

目的探索微孔板法药物敏感性试验（DST）和全基因组测序（WGS）检测4种常用抗结核药物耐药性在儿科临床应用价值。方法复苏培养阳性的结核分枝杆菌（MTB）保存菌株61株，分别行比例法、微孔板法和WGS检测异烟肼、利福平、乙胺丁醇和链霉素的耐药性。使用一致性检验检测微孔板法和WGS与比例法的一致性。结果 61株MTB菌株经比例法检测出48株（78.7%）全敏感菌株，13株（21.3%）对至少1种药物耐药，对4种药物共同耐药的3株（4.9%）。以比例法DST为金标准，微孔板法检测异烟肼耐药的敏感度、特异度和Kappa值分别为100.0%、92.0%和0.81，利福平分别为85.7%、98.2%和0.84，乙胺丁醇分别为0.0%、100.0%和0.00，链霉素分别为80.0%、98.0%和0.81;WGS检测异烟肼耐药的敏感度、特异度和Kappa值分别为90.9%、94.0%和0.79，利福平分别为100%、98.2%和0.92，乙胺丁醇分别为60.0%、94.6%和0.50，链霉素分别为80.0%、98.0%和0.81。结论微孔板法和WGS对异烟肼、利福平和链霉素耐药检测均有较高的敏感度与特异度，与比例法结果高度一致，均可应用于临床。而对乙胺丁醇耐药性检测的一致性较差，建议使用比例法结合WGS结果来综合评判MTB乙胺丁醇的耐药性。

关键词: 全基因组测序, 微孔板, 一致性检验, 最小抑菌浓度, 儿童

Abstract:

Objective To explore the value of drug susceptibility test (DST) by micropore-plate method and whole-genome sequencing (WGS) in detecting the drug resistance of anti-tuberculosis drugs in children. Methods Sixty-one partially preserved strains with positive culture of Mycobacterium tuberculosis (MTB) were collected. The drug resistance of isoniazid, rifampicin, ethambutol, and streptomycin were detected by proportional method, micropore-plate method DST and WGS. The variants of four drug resistance related genes in WGS were summarized, and the clinical data, treatment plan and recovery of the affected children were collected. The reasons for the differences of drug resistance detected by the three methods were analyzed. Results Among 61 MTB strains, 78.69% (n=48) were pan-susceptible strains, 1.64% (n=1) was isoniazid-resistant strain, 1.64% (n=1) was streptomycin resistant strain, 6.56% (n=4) were isoniazid and streptomycin resistant strains, 3.28% (n=2) were isoniazid, rifampicin, and ethambutol resistant strains, 3.28% (n=2) were isoniazid, rifampicin and streptomycin resistant strains, and 4.92% (n=3) were resistant to the four drugs. The sensitivity, specificity, positive predictive value, negative predictive value, and the Kappa value of micropore-plate method for predicting isoniazid resistance were 100%, 92.0%, 73.3%, 100% and 0.81, for rifampicin were 85.7%, 98.2%, 85.7%, 98.2% and 0.84, for ethambutol were 0.0%, 100%, 0.0%, 91.8% and 0.0, for streptomycin were 80.0%, 98.0%, 88.9%, 96.2% and 0.81, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, and the Kappa value of WGS for predicting isoniazid resistance were 90.9%, 94.0%, 76.9%, 97.9% and 0.79, for rifampicin were 100%, 98.2%, 87.5%, 100% and 0.92, for ethambutol were 60.0%, 94.6%, 50.0%, 96.4% and 0.50, for streptomycin were 80.0%, 98.0%, 88.9%, 96.2% and 0.81, respectively. Conclusion Highly consistent with ratio method, micropore-plate method and WGS have higher sensitivity to predict isoniazid, rifampin, and streptomycin resistant tuberculosis in children, which can replace traditional proportional method in clinical practice. However, it is suggested to use proportional method combined with WGS to comprehensively evaluate the ethambutol resistance of MTB, as the consistency of micropore-plate and ratio methods is poor.

Key words: whole genome sequencing, micropore-plate method, consistency test, minimum inhibitory concentration, child

张颖, 任巧丽, 赵瑞秋, 许红梅, 龙晓茹. 儿童抗结核药物耐药比例法、微孔板法、全基因组测序检测对比研究[J]. 临床儿科杂志, 2023, 41(2): 117-124.

ZHANG Ying, REN Qiaoli, ZHAO Ruiqiu, XU Hongmei, LONG Xiaoru. Comparison of the ratio method drug susceptibility test, micropore-plate method and whole-genome sequencing of anti-tuberculosis drugs in children[J]. Journal of Clinical Pediatrics, 2023, 41(2): 117-124.

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基因	变异位点	比例法			MIC/μg·mL^-1
基因	变异位点	INH耐药(n=11)	INH敏感(n=50)	P¹⁾	≥1.6(n=15)	0.2(n=46)	P¹⁾
katG	Ser140Asn	0(0.0)	1(2.0)	1.000	1(6.7)	0(0.0)	0.246
	Ser315Thr	9(81.8)	2(4.0)	<0.001	11(73.3)	0(0.0)	<0.001
fabG1	-15C>T	1(9.1)	0(0.0)	0.180	1(6.7)	0(0.0)	0.246
inhA	Ile194Thr	1(9.1)	0(0.0)	0.180	1(6.7)	0(0.0)	0.246

基因	变异位点	比例法			MIC/μg·mL^-1
基因	变异位点	RFP耐药(n=7)	RFP敏感(n=54)	P¹⁾	>2.0(n=7)	0.5(n=4)	0.3(n=50)	P¹⁾
rpoB	Ser450Leu	4(57.1)	0(0.0)	<0.001	4(57.1)	0(0.0)	0(0.0)	<0.001
	His445Asn	1(14.3)	0(0.0)	0.115	0(0.0)	1(25.0)	0(0.0)	0.066
	His445Asp	1(14.3)	0(0.0)	0.115	1(14.3)	0(0.0)	0(0.0)	0.180
	His445Leu	0(0.0)	1(1.9)	1.000	1(14.3)	0(0.0)	0(0.0)	0.180
	His445Tyr	1(14.3)	0(0.0)	0.115	1(14.3)	0(0.0)	0(0.0)	0.180

基因	变异位点	比例法			MIC/μg·mL^-1
基因	变异位点	EMB耐药(n=5)	EMB敏感(n=56)	P¹⁾	5.0(n=2)	2.5(n=59)	P¹⁾
embB	Ala356Val	0(0.0)	1(1.8)	1.000	0(0.0)	1(1.7)	1.000
	Gln497Arg	1(20.0)	0(0.0)	0.082	0(0.0)	1(1.7)	1.000
	Gly406Ser	0(0.0)	1(1.8)	1.000	0(0.0)	1(1.7)	1.000
	Met306Ile	1(20.0)	0(0.0)	0.082	0(0.0)	1(1.7)	1.000
	Met306Val	1(20.0)	1(1.8)	0.378	2(100.0)	0(0.0)	0.001

基因	变异位点	比例法			MIC/μg·mL^-1
基因	变异位点	SM耐药(n=10)	SM敏感(n=51)	P¹⁾	≥8.0(n=8)	4.0(n=1)	1.0(n=52)	P¹⁾
rpsL	Lys43Arg	6(60.0)	1(2.0)	<0.001	7(87.5)	0(0.0)	0(0.0)	<0.001
	Lys88Arg	1(10.0)	0(0.0)	0.164	0(0.0)	1(100.0)	0(0.0)	0.016
rrs	514a>c	1(10.0)	0(0.0)	0.164	1(12.5)	0(0.0)	0(0.0)	0.148

鉴定方法	耐药属性	治疗成功组(n=55)	治疗失败或死亡组(n=6)	χ²值	P
比例法	INH耐药	10(18.2)	1(16.7)	0.00	1.000
	RFP耐药	6(10.9)	1(16.7)	0.00	1.000
	EMB耐药	4(7.3)	1(16.7)	0.00	0.990
	SM耐药	10(18.2)	0(0.0)	-	0.577¹⁾
微孔板法	INH耐药	14(25.5)	1(16.7)	0.00	1.000
	RFP耐药	5(9.1)	2(33.3)	1.20	0.274
	SM耐药	9(16.4)	0(0.0)	-	0.579¹⁾
WGS	INH耐药	12(21.8)	1(16.7)	0.00	1.000
	RFP耐药	6(10.9)	2(33.3)	0.83	0.364
	EMB耐药	5(9.1)	1(16.7)	0.00	1.000
	SM耐药	9(16.4)	0(0.0)	-	0.579¹⁾