药物敏感性试验与全基因组测序检测抗结核药物耐药性的比较研究

Comparative study of drug susceptibility testing and whole genome test testing anti-tuberculosis drug resistance

目的通过药物敏感性试验(DST)和全基因组测序(WGS)检测结核分枝杆菌(MTB)的耐药情况,比较二者一致性,探讨全基因测序用于MTB耐药性检测的特点。方法选取2018-2020年留存于四川大学华西医院共71例MTB临床分离株,采用DST(氧化还原指示剂法)和WGS两种方法对菌株进行异烟肼(INH)、利福平(RIF)、链霉素(SM)、乙胺丁醇(EMB)、莫西沙星(MFX)、氧氟沙星(OFX)、左氧氟沙星(LFX)、卡那霉素(KAN)、阿米卡星(AMK)、卷曲霉素(CPM)、乙硫异烟胺(ETH)、利福布汀(RFB)、对氨基水杨酸(PAS)和氯法齐明(CLO)共14种药物的耐药性检测,并对结果进行Kappa检验分析。结果两种方法对RIF、RFB、SM、MFX、OFX和LFX 6种药物耐药性检测的符合率均超过90.00%,Kappa值均大于0.80,一致性较好;INH和EMB耐药性检测的符合率分别为84.51%,81.69%,Kappa值分别为0.68和0.54,一致性一般;AMK和KAN两种方法检测的耐药株未超过2株,耐药率小于3.00%;CPM、ETH、PAS、CLO耐药性检测的符合率范围为61.97%~91.55%,Kappa值均小于0.40,一致性较差。结论WGS检测不同抗结核药物耐药性的能力存在差异,其检测RIF、RFB、SM、MFX、OFX和LFX 6种药物耐药性效果较佳,而检测其他抗结核药物耐药性与DST相比较仍存在一定差异,需要进一步明确相关药物的详细耐药机制,并对WGS用于耐药性分析的标准化进行探索。

Objective To compare the categorical agreement between drug susceptibility testing(DST)and whole genome sequencing(WGS)for the detection of drug resistance in Mycobacterium tuberculosis(MTB),and to explore the characteristics of WGS for MTB drug resistance detection.Methods A total of 71 MTB clinical isolates retained in West China Hospital of Sichuan University from 2018 to 2020 were included in this study.The MTB strains were tested for resistance to 14 anti-tuberculosis drugs,including Isoniazid(INH),Rifampicin(RIF),Rifabutin(RFB),Ethambutol(EMB),Streptomycin(SM),Moxifloxacin(MFX),Ofloxacin(OFX),Levofloxacin(LFX),Amikacin(AMK),Kanamycin(KAN),Capreomycin(CPM),Para-aminosalicylic acid(PAS),Ethionamide(ETH)and Clofazimine(CLO),using both DST(colorimetric redox indicator method)and WGS methods.Kappa test was performed to analyze the results of drug resistance detection for both methods.Results Based on DST and WGS methods to detect anti-tuberculosis drug resistance in seventy-one MTB clinical isolates,the results showed that the agreement rate of RIF,RFB,SM,MFX,OFX and LFX exceeded 90.00%,and the kappa values were all greater than 0.80,with near perfect agreement;The agreement rates of INH and EMB were 84.51%and 81.69%,and Kappa values were 0.68 and 0.54,respectively,with fair agreement.No more than two drug resistant MTB strains of AMK and KAN were detected by both methods,and the resistance rate was less than 3.00%.The agreement rates of CPM,ETH,P AS,and CLO ranged from 61.97%to 91.55%,and the Kappa values were less than 0.40,with slight or fair agreement.Conclusion There are differences in the ability of WGS to detect resistance to various anti-tuberculosis drugs,and it is more effective in detecting resistance to six anti-tuberculosis drugs,including RIF,RFB,SM,MFX,OFX and LFX,while there are still certain differences in detecting resistance to other anti-tuberculosis drugs compared with DST.It is necessary to further clarify the detailed resistance mechanisms of relevant anti-tuberculosis drugs and to explore the standardization of WGS for drug resistance detection.