江西省结核分枝杆菌耐二线抗结核药的分子学特征

目的 初步明确江西省耐二线抗结核药相关基因突变的特征，评价等位基因特异性多重PCR（multiplex allele-specific polymerase chain reaction，MAS-PCR）检测二线抗结核药耐药性的可行性。 方法 采用DNA测序方法和MAS-PCR技术，对江西省52株耐二线抗结核药的结核分枝杆菌进行耐药相关基因突变位点检测。 结果DNA测序分析：39株耐氧氟沙星（Ofx）菌株中，32株存在gyrA基因错义突变，2株发生gyrB基因错义突变。29株耐卡那霉素（Km）或卷曲霉素（Cm）菌株中，22株为rrs1401位点A→G突变。52株耐二线抗结核药的结核分枝杆菌，40株为北京基因型（76.92%，40/52），其中17株北京基因型菌株发生gyrA-GAC94GGC突变（42.50%，17/40），19株北京基因型菌株发生rrs-A1401G突变（47.50%，19/40）。北京基因型与基因突变类型gyrA-GAC94GGC和rrs-A1401G无明显相关性（χ²=1.16、1.92，P值均＞0.05）。MAS-PCR检测Ofx耐药株的敏感度为61.54%（24/39），检测Km或Cm耐药株的敏感度为79.31%（23/29）。 结论 gyrA基因94位密码子突变是江西省结核分枝杆菌Ofx耐药的主要机制; rrs-A1401G突变则是Km或Cm耐药的主要原因。MAS-PCR方法对于快速检测二线抗结核药的耐药性有一定的临床应用价值。

Molecular characteristics of the second-line antituberculosis drug-resistant Mycobacterium tuberculosis strains from Jiangxi province

Objective To initially ascertain molecular characteristics of the second-line antituberculosis drug- resistant M. tuberculosis clinical isolates in Jiangxi, and to evaluate the feasibility of multiplex allele-specific poly- merase chain reaction （MAS-PCR） assay for the detection of the second-line drug resistance in M. tuberculosis clini- cal isolates. Methods Fifty-two second-line drug-resistant isolates and 30 pan-susceptible isolates from Jiangxi province were analyzed for gene mutations related to the second-line drug resistance using MAS-PCR and DNA se- quencing. Results DNA sequencing indicated that 32 of 39 ofloxacin （Ofx）-resistant isolates displayed missense mutations in gyrA gene, and the most common mutations were observed at codon 94 （n= 27）, and only 2 isolates showed single-point mutation in gyrB gene. In addition, 22 of 29 kanarnycin （Kin）- or capreomycin （Cm）-resistant isolates had an A-to-G transition at nucleotide position 1401 of rrs gene. It is shown that the Beijing genotype was predominant （76.92%, 40/52） among the 52 drug-resistant strains. Of 40 Beijing genotype strains, 17 strains （42.50%）displayed gyrA-GAC94GGC mutations, and 19 strains （47.50%） showed rrs-A1401G mutations. No re- lationship could be demonstrated between Beijing genotype and gyrA-GAC94GGC or rrs-A1401G mutations（X^2= 1.16,1.92, P value〉0. 05）. In comparison with the phenotypic data, the sensitivities of the detection of Ofx resist- iance, and Km or Cm resistance using MAS-PCR were 61.54%（24/39）and 79. 31%（23/29） respectively. Conclusion In Jiangxi province, gyrA mutation is the main molecular mechanism of Ofx resistance in M. tuberculosis, and an A1401G mutation in the rrs gene is the main cause of Km or Cm resistance. MAS-PCR, an assay with technical sim-olicity, short turnaround time, and low cost, could be useful for screening second-line drug-resistant M. tuberculosis, particularly in resource-limited areas with a high prevalence of tuberculosis and drug resistance.