青海地区结核分枝杆菌异烟肼耐药相关基因突变特征

目的探讨青海地区耐异烟肼结核分枝杆菌的基因突变特征。方法收集198株结核菌,采用比例法检测这些菌株对一线抗结核药物异烟肼(INH)、利福平、链霉素、乙胺丁醇的表型耐药情况,PCR扩增INH耐药相关基因,进行基因测序并分析结果。结果在198株表型耐药受试结核菌中,对INH、利福平、链霉素和乙胺丁醇耐药的菌株分别占45.96%(91/198)、43.94%(87/198)、40.91%(81/198)和30.30%(60/198)。在对其中89株INH耐药和107株INH敏感的共计196株进行的INH耐药相关基因测序分析中,katG315位点、oxyR-ahpC间隔区和pre-inhA基因突变率分别为30.10%(59/196)、9.69%(19/196)和3.57%(7/196)。其中,katG315位点突变率在耐药菌株中为61.80%(55/89),在敏感株中为3.74%(4/107);oxyR-ahpC间隔区突变率在耐药株中为19.10%(17/89),在敏感株中为1.87%(2/107);pre-inhA突变率在突变株中为3.37(3/89),在敏感株中为3.74%(4/107)。结果表明,INH耐药株的katG315位点和oxyR-ahpC间隔区突变率远高于INH敏感菌株,有显著的统计学差异(χ^2=75.105,P<0.001;χ^2=13.456,P<0.001),而INH耐药株的pre-inhA突变率与敏感株相比则无显著差异(χ^2=0.019,P=0.890);katG基因突变检测异烟肼耐药的敏感度为66.29%,特异度为93.46%。结论青海地区结核分枝杆菌的INH耐药率较高,其INH耐药与katG315位点高突变率相关;检测katG位点、oxyR-ahpC间隔区和pre-inhA的基因突变情况可供判断结核分枝杆菌的异烟肼耐药。

Characteristics of the gene mutations related to the isoniazid-resistance of Mycobacterium tuberculosis from Qinghai area

Objective To explore gene mutation characteristics of the isoniazid(INH)-resistant Mycobacterium tuberculosis from Qinghai area. Methods In total 198 strains of M. tuberculosis were collected in the Qinghai area,and the phenotypic resistance was determined by the proportional method. The genes related to the INH resistance were amplified by PCR and then sequenced. Results In the tested 198 strains of M. tuberculosis,the strains resistant to the antituberculosis drugs,INH,rifampicin(RFP),streptomycin(SM)and ethambuto(EMB)l,were 45.96%(91/198)for INH,43.94%(87/198)for RFP,40.91%(81/198)for SM and30.30%(60/198)for EMB of total strains,respectively. Among them,a total of 196 strains,including the 89 INH resistant and 107 INH sensitive strains,were subject to the sequencing of the genes related to the INH resistance. A total mutation rate of the katG315 site,oxyR-ahpC intergenic region and pre-inhA genes in the 196 strains was 30.10%(59/196),9.69%(19/196)and 3.57%(7/196),respectively. In addition,the mutation rate of katG315 site was 61.80%(55/89)in INH resistant strains and 3.74%(4/107)in INH sensitive strains. The mutation rate of oxyR-ahpC intergenic region was 19.10%(17/89)in INH resistant strains and 1.87%(2/107)in INH sensitive strains. The mutation rate of pro-inhA was 3.37%(3/89)in INH resistant strains and 3.74%(4/107)in INH sensitive strains. The statistical analyses indicated that the occurrence of the katG315 and oxyR-ahpC intergenic region mutations was significantly higher in INH resistant strains than that in sensitive ones(χ^2=75.105,P<0.001;χ^2=13.456,P<0.001). However,the preinhA gene mutations were not statistically significant between the INH resistant and sensitive isolates(χ^2=0.019,P=0.890). The sensitivity of katG gene mutation for isoniazid resistance detection was 66.29%,and the specificity was 93.46%. Conclusion The INH resistant strains were found in a much higher rate in M. tuberculosis isolates from Qinghai area,which was related to the higher rate of the katG gene mutations. Detection of the katG,oxyR-ahpC intergenic region and pre-inhA gene mutations could be useful for diagnosing the INH resistance of M. tuberculosis.