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基于全基因组测序的2022－2023年河北省耐多药结核分枝杆菌耐药突变特征分析及检测效能评价

Multidrug-resistant Mycobacterium tuberculosis drug resistance mutation characteristics and detection based on whole genome sequencing in Hebei, 2022−2023

    摘要
  • 摘要:
    目的  了解河北省耐多药结核分枝杆菌（MDR-MTB）耐药相关基因突变特征，评估全基因组测序（WGS）的耐药检测效能，验证WGS在结核病临床诊疗和耐药监测中的应用潜能。
    方法  收集2022－2023年河北省11个市的结核分枝杆菌临床分离株，进行菌种鉴定和比例法表型药敏试验（pDST）后，对筛选出的152株MDR-MTB菌株进行WGS分析。 与H37Rv参考基因组序列进行比对后获得耐药突变位点，以pDST结果为金标准，评价WGS对10种抗结核药物耐药性预测的灵敏度、特异度、一致性等。
    结果  152株MDR-MTB分离株对异烟肼（INH）、利福平（RFP）、链霉素（SM）、乙胺丁醇（EMB）、氟喹诺酮类（FQs）、二线注射类SLIDs，包括卡那霉素（KM）、阿米卡星（AM）、卷曲霉素（CM）、吡嗪酰胺（PZA）的耐药相关基因的主要突变类型为katG p.Ser315Thr（115/143，80.42%）、rpoB p.Ser450Leu（112/151，74.17%）、rpsL p.Lys43Arg（108/136，79.41%）、embB p.Met306Val（32/83，38.55%）、gyrA p.Ala90Val（24/65，36.92%）、rrs n.1401A>G（93.33%~100.00%）、pncAp.Asp136Gly（12/70，17.14%）；此外，其中1株分离株出现利奈唑胺（LZD）耐药相关基因突变，突变位点为RplC p.Cys154Arg。 WGS预测MDR-MTB对INH和RFP耐药性的灵敏度分别为94.08%和99.34%；预测其对莫西沙星、氧氟沙星、左氧氟沙星耐药性的灵敏度为95.31%~96.77%，特异度为93.41%~95.45%；预测其对KM和AM耐药性的特异度均为100.00%。
    结论  WGS预测INH、RFP、FQs、KM、AM、CM耐药性表现良好，在结核病诊疗和耐药监测中具有较高的应用潜力。

     

    Abstract:
    Objective  To understand the drug resistance-associated gene mutation characteristics of multidrug-resistant Mycobacterium tuberculosis (MDR-MTB), evaluate the efficacy of whole-genome sequencing (WGS) in the detection of drug resistance of MDR-MTB in Hebei province, and verify the application potential of WGS in clinical tuberculosis (TB) diagnosis and drug resistance surveillance.
    Methods Clinical isolates of M. tuberculosis were collected from 11 cities in Hebei between 2022 and 2023. After species identification and phenotypic drug susceptibility testing (pDST) with proportion method, 152 MDR-MTB isolates were subjected to WGS. The drug resistance mutation loci were identified by alignment with the genome sequence of H37Rv, and the sensitivity, specificity, and consistency of WGS in the detection of the resistance of MDR-MTB to 10 anti-TB drugs were evaluated by using pDST results as the gold standard.
    Results  In the Results In the 152 MDR-MTB isolates, the primary resistance-associated mutation types to isoniazid (INH), rifampicin (RFP), streptomycin (Sm), ethambutol (EMB), fluoroquinolones (FQs), the second-line injectable drugs (SLIDs), including kanamycin (KM), amikacin (AM), and capreomycin (CM), and pyrazinamide (PZA) were katG p.Ser315Thr (115/143, 80.42%), rpoB p.Ser450Leu (112/151, 74.17%), rpsL p.Lys43Arg (108/136, 79.41%), embB p.Met306Val (32/83, 38.55%), gyrA p.Ala90Val (24/65, 36.92%), rrs n.1401A>G (14/15-14/14, 93.33%~100.00%) and pncA p.Asp136Gly (12/70, 17.14%). Additionally, one isolate exhibited mutation associated with resistance to linezolid (LZD) at locus RplC p.Cys154Arg. The sensitivities of WGS for predicting resistance of MDR-MTB to INH and RFP were 94.08% and 99.34%, respectively.The sensitivities for predicting resistance to moxifloxacin, ofloxacin, and levofloxacin ranged from 95.31% to 96.77%, with specificities ranging from 93.41% to 95.45%.the specificities for predicting resistance to KM and AM were both 100.00%.
    Conclusion  WGS demonstrated good performance in the detection of the resistance of MDR-MTB to INH, RFP, FQs, KM, AM, and CM, indicating high potential for the application in TB diagnosis and drug resistance surveillance.

     

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