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中国防痨杂志 ›› 2022, Vol. 44 ›› Issue (2): 169-173.doi: 10.19982/j.issn.1000-6621.20210445

• 论著 • 上一篇    下一篇

结核分枝杆菌利福平基因型药敏和表型药敏检测结果不一致原因分析

王少华, 赵国连(), 王佩, 谈小文, 崔晓利, 康磊, 党丽云   

  1. 西安市胸科医院,西安 710100
  • 收稿日期:2021-08-04 出版日期:2022-02-10 发布日期:2022-02-14
  • 通信作者: 赵国连 E-mail:774567495@qq.com
  • 基金资助:
    西安市创新能力强基计划-医学研究项目(21YXYJ0001)

Analysis of inconsistency between genotypic and phenotypic results of Mycobacterium tuberculosis rifampicin susceptibility test

WANG Shao-hua, ZHAO Guo-lian(), WANG Pei, TAN Xiao-wen, CUI Xiao-li, KANG Lei, DANG Li-yun   

  1. Xi’an Chest Hospital, Xi’an 710100,China
  • Received:2021-08-04 Online:2022-02-10 Published:2022-02-14
  • Contact: ZHAO Guo-lian E-mail:774567495@qq.com
  • Supported by:
    Xi’an Innovation Ability Base Plan-Medical Research Project(21YXYJ0001)

摘要:

目的: 探讨荧光PCR探针熔解曲线法(简称“熔解曲线法”)与微孔板法检测利福平药物敏感性试验(简称“药敏试验”)结果不一致的原因,为临床基因型与表型药敏结果不一致提供解释。 方法: 收集2019年8月至2020年9月西安市胸科医院收治的分枝杆菌培养阳性的结核病患者资料,共2562例,筛选同时进行利福平熔解曲线法耐药基因检测和微孔板法表型药敏试验且结果不一致的菌株1294株。通过对rpoB基因测序,分析不一致结果与rpoB基因突变位点的相关性。 结果: 54例(4.17%,54/1294)熔解曲线法与微孔板法检测结果不一致的患者中,45例为熔解曲线法检测突变型而微孔板法检测敏感,8例为熔解曲线法检测野生型而微孔板法检测耐药,1例熔解曲线法检测出异质性耐药,未纳入分析。其中rpoB基因在507~512位密码子发生突变时,其与微孔板法结果不一致率最高,为70.59%(24/34),且不一致的菌株在微孔板法中最低抑菌浓度(MIC)的测定值多分布在≤1μg/ml。Leu511Pro是观察到不一致菌株最多的突变位点,占45.28%(24/53),其次观察到较多的突变位点是Leu533Pro,占所有不一致菌株的15.09%(8/53),Asp516Tyr 和His526Asn突变率均为5.66%(3/53)。8例熔解曲线法检测敏感而微孔板法检测耐药的菌株进行测序,结果显示rpoB基因区域未发生突变。结论: rpoB基因Leu511Pro、Leu533Pro、Asp516Tyr和His526Asn位点突变是造成临床结核分枝杆菌基因型与表型药敏结果不一致的主要原因,但其是否与低水平耐药机制有关还需要进一步研究。

关键词: 分枝杆菌,结核, 抗药性,细菌, 微生物敏感性试验, 数据说明,统计

Abstract:

Objective: To investigate the inconsistency between the fluorescent PCR probe melting curve method (“melting curve method”) and the microplate method in detecting the susceptibility of rifampicin, and to provide an explanation for the inconsistency between clinical genotypic and phenotypic drug susceptability test (pDST) results. Methods: We collected the data of 2562 culture positive tuberculosis patients in Xi’an Chest Hospital from August 2019 to September 2020 and screened out 1294 strains with different results using melting curve method and microplate pDST. The correlation between the inconsistent results and the mutation of rpoB gene were analyzed by sequencing the rpoB gene. Results: Among the 54 patients (4.17%, 54/1294) whose melting curve testing results were inconsistent with the pDST test results, 45 were melting curve method positive for mutants but pDST negative, and 8 were melting curve method negative for mutants but pDST positive. One case was detected as having heterogeneous drug resistance by melting curve method, thus was excluded from the analysis. When the rpoB gene was mutated at codons of 507-512, the inconsistency rate with the pDST results was the highest (70.59%, 24/34), and the minimum inhibitory concentrations of the inconsistent strains tested with the microplate method were mostly ≤1 ug/ml. Leu511pro was the most frequently observed mutation, accounting for 45.28% (24/53), followed by Leu533pro, accounting for 15.09% (8/53) of all of the inconsistent strains. Both Asp516Tyr and His526Asn mutations accounted for 5.66% (3/53). All 8 strains that were melting curve method negative for mutants but pDST positive were sequenced as no mutation in the rpoB gene region. Conclusion: The mutations of Leu511Pro, Leu533Pro, Asp516Tyr and His526Asn in the rpoB region were the main reasons for the inconsistency between the genotypic and phenotypic results for Mycobacterium tuberculosis susceptibility to rifampicin, but whether it was related to the low-level drug resistance mechanism still need further research.

Key words: Mycobacterium tuberculosis, Drug resistance,bacterial, Microbial sensitivity tests, Data interpretation,statistical

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