浙江省结核分枝杆菌耐药突变特征及其与基因型相关性分析

目的: 评价全基因组测序(whole-genome sequencing,WGS)对浙江省结核分枝杆菌(Mycobacterium tuberculosis,MTB)耐药相关基因突变特征及其与基因型相关性的耐药分析和检测效能。方法: 对2018—2019年浙江省第五次结核病耐药监测项目收集的808株MTB菌株进行14种抗结核药物的WGS分析。鉴定菌株的基因型及耐药相关基因突变,并对二者相关性,以及WGS检测异烟肼的药物敏感性试验(简称“药敏试验”)结果与表型药敏试验结果的一致性进行分析。结果: 808株MTB菌株中,153株对11种抗结核药物(异烟肼、利福平、乙胺丁醇、吡嗪酰胺、链霉素、氟喹诺酮类、阿米卡星、卡那霉素、卷曲霉素、乙硫异烟胺、对氨基水杨酸)发生了耐药相关基因突变,总突变率为18.9%。主要耐药突变类型分别为katG-315-S/T异烟肼,60.0%(45/75)]、rpoB-450-S/L利福平,57.1%(16/28)]、embB-306-M/V乙胺丁醇,43.8%(7/16)]、rpsL-43-K/R链霉素,65.5%(36/55)]、gyrA-94-D/G氟喹诺酮类,36.6%(15/41)]、rrs-1402-C/A(阿米卡星,3/3)、rrs-1402-C/A(卡那霉素,3/4)、rrs-1402-C/A(卷曲霉素,3/3)、inhA-15-C/T乙硫异烟胺,65.0%(13/20)]、thyA-75-H/N(对氨基水杨酸,7/8)。rpsL基因和katG-315位点在北京基因型中的耐药突变率均为6.8%(40/586)]均明显高于二者在非北京基因型中的突变率0.0%(0/222)和3.2%(7/222)],差异均有统计学意义(χ²=15.943,P=0.000;χ²=3.964,P=0.046)。WGS耐药性分析对于异烟肼的检测敏感度、特异度、阳性预测值、阴性预测值、一致率和Kappa值分别为87.5%(49/56)、98.0%(680/694)、77.8%(49/63)、99.0%(680/687)、97.2%(729/750)和0.808。结论: 浙江省MTB菌株对11种抗结核药物耐药相关基因突变以katG、rpoB、embB、pncA、rpsL、gyrA、rrs、thyA、inhA基因上的突变为主,其中rpsL基因、katG-315和rpsL-43位点突变与北京基因型相关。WGS耐药性分析对于异烟肼具有全面的耐药检测性能,但对个别基因突变位点的检测性能较弱。

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

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.