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基于全基因组测序的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

Analysis of Drug Resistance Mutation Characteristics and Evaluation of Detection Efficacy of Multidrug-Resistant Mycobacterium Tuberculosis in Hebei Province Based on Whole Genome Sequencing

    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2022一2023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（14/15−14/14，93.33%−100.00%）、pncA_p.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预测不同抗结核药物耐药性的灵敏度和特异度分别为INH：94.08%，－；RFP：99.34%，－；Sm：97.10%，78.57%；EMB：76.81%，63.86%；Mfx：95.31%，95.45%；Ofx：96.77%，94.44%；Lfx：96.72%，93.41%；Km：88.24%，100.00%；Am：93.33%，100.00%；Cm：81.25%，98.53%。
    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 investigate the drug resistance-associated gene mutation characteristics of multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) strains in Hebei Province, evaluate the efficacy of whole-genome sequencing (WGS) in detecting drug resistance, and verify the potential application of WGS in clinical diagnosis and drug resistance surveillance of tuberculosis.
    Methods  Clinical isolates of Mycobacterium tuberculosis were collected from 11 cities in Hebei Province between 2022 and 2023.. After species identification and phenotypic drug susceptibility testing (pDST) using the proportion method, 152 MDR-MTB isolates were subjected to WGS analysis. The drug resistance mutation sites were identified by aligning with the genome sequence of H37Rv, and the sensitivity, specificity, and consistency of WGS for predicting resistance to 10 anti-tuberculosis drugs were evaluated using pDST results as the gold standard.
    Results  Among the 152 MDR-MTB isolates, the primary resistance-associated mutation types to isoniazid (INH), rifampicin (RFP), streptomycin (Sm), ethambutol (EMB), fluoroquinolones (FQs), 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 a mutation associated with resistance to linezolid (Lzd), specifically RplC_p.Cys154Arg. The sensitivity and specificity of WGS in predicting resistance to different anti-tuberculosis drugs were as follows: INH: 94.08%, –; RFP: 99.34%, –; Sm: 97.10%, 78.57%; EMB: 76.81%, 63.86%; Mfx: 95.31%, 95.45%; Ofx: 96.77%, 94.44%; Lfx: 96.72%, 93.41%; Km: 88.24%, 100.00%; Am: 93.33%, 100.00%; Cm: 81.25%, 98.53%.
    Conclusion  WGS demonstrated good performance in predicting resistance to INH, RFP, FQs, Km, Am, and Cm, indicating high potential for application in tuberculosis diagnosis and drug resistance surveillance.

     

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