Genomic mutations in the katG, inhA and aphC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa.

Genotypic analysis of isoniazid (INH) resistance in 79 isolates of M. tuberculosis (MTB) was undertaken by PCR-single strand conformation polymorphism (SSCP), Msp1 restriction enzyme analysis and sequence analysis of specific regions of three genes (part of the coding sequence of katG, and promoter regions of the inhA operon and ahpC) in order to determine the particular allelic variants within these genes. The epidemiologic relatedness was determined using IS6110 and polymorphic G-C region (PGRS (MTB484(1)) based restriction fragment length polymorphism (RFLP). Mutations in katG, inhA locus and ahpC were identified in 77/79, 19/79 and 10/79 isolates respectively. The ability of PCR-SSCP to detect mutations associated with INH resistance in katG, inhA and ahpC genes was 100% (CI 91.2-99.7%), 98.7% (CI 74.0-99.9%), and 100% (CI 69.2-100%) respectively. Specificity was 100%. All isolates with mutations in the 209 bp fragment of the MTB katG gene containing the Ser315Thr codon were positive by PCR-RFLP using Msp1 enzyme restriction analysis. Sixteen of 19 isolates with alterations on the 3' end of the ribosome binding site upstream of mabA in inhA locus simultaneously harbored Ser315Thr mutations in KatG. In 9/10 isolates, mutations in the ahpC promoter region were located in the 105 bp oxyR-ahpC intergenic region. None of 17 INH drug susceptible isolates harbored mutations in any of the three genetic regions, although the katG1 allele (Arg 463 Leu) was present in one isolate. Characterization by IS6110/PGRS(MTB484(1))RFLP analysis revealed that a number of drug resistant clones are widespread in the community. We conclude that the frequency of the Ser315Thr katG mutation in the local strain population makes the PCR-RFLP MTB katG assay a reliable, rapid and useful method for detecting INH resistance.     

耐异烟肼结核分枝杆菌临床分离株耐药相关基因突变研究

目的阐明结核分枝杆菌耐异烟肼临床分离株katG、inhA、ahpC、kasA及oxyR基因突变特点。方法对144株结核分枝杆菌临床分离株(耐异烟肼菌株101株;异烟肼敏感株43株)的katG、inhA、kasA、ahpC及oxyR基因进行DNA片断扩增及DNA序列分析,与GeneBank中结核分枝杆菌标准序列进行比较。结果(1)耐异烟肼菌株中未发现katG完全缺失,81株耐药株(80.2%)katG存在点突变、缺失或插入,其中16个突变位点未见报道;39株(38.6%)耐药株第315位点突变,低耐药菌株(1μg/ml)第315位点突变率显著高于高耐药菌株(10μg/ml;χ2=9.31,P<0.05);58株(57.4%)耐药株第463位点突变。23株(53.3%)敏感株第463位点突变。(2)5株(4.9%)耐药株inhA发生突变。敏感株inhA无突变。(3)3株(2.9%)耐药株ahpC发生突变。敏感株ahpC无突变。(4)17株(16.8%)耐药株kasA发生突变。敏感株中3株菌株Gly312Ser突变。(5)在全部菌株中未发现oxyR基因突变。(6)综合本项研究中各基因的突变情况,共有91株耐异烟肼菌株发生与异烟肼耐药相关的突变。结论本项研究进一步证实了结核分枝杆菌耐异烟肼与katG、inhA、ahpC及kasA基因突变之间的关系,并且提示还有其他机制参与异烟肼耐药。     

福建省结核分枝杆菌异烟肼耐药相关基因突变特征初步分析

目的 了解福建省结核分枝杆菌异烟肼耐药相关基因的突变特征,为异烟肼耐药快速检测方法的建立提供一定的科学依据。方法 对来源于福建省结核病耐药性监测30个监测点纳入的75株耐多药和10株全敏感结核分枝杆菌分离株,进行katG、inhA、oxyR-ahpC基因片段PCR扩增并测序分析,用RD105缺失基因检测法进行北京家族基因型鉴定,使用卡方检验分析相关性。结果 10株全敏感株未检测到突变。75株耐多药结核分枝杆菌检测到72株katG、inhA、oxyR-ahpC发生单一或联合基因突变,突变率为96.0%(72/75)。其中,65株(86.7%,65/75)发生katG突变,涉及5个位点,最常见位点突变的密码子是315,突变率为82.7 %(62/75),最常见突变形式为Ser315Thr(77.3%,58/75);8株(10.7%,8/75)发生inhA突变,突变形式均为C(-15)T;5株(6.7%,5/75)发生oxyR-ahpC突变,突变形式为C(-39)T或C(-46)A。katG、inhA和oxyR-ahpC 在北京家族基因型菌株和非北京家族基因型菌株中的突变率分别为83.9 %(47/56)、12.5%(7/56)、7.1 %(4/56)和94.7 %(18/19)、5.3 %(1/19)、5.3 %(1/19),差异无统计学意义(P值分别为0.23、0.38、0.78)。结论 福建省结核分枝杆菌异烟肼耐药性相关基因突变绝大多数发生在katG、inhA和oxyR-ahpC基因位点,且以katG突变为主。初步分析显示北京家族基因型菌株流行与异烟肼耐药基因突变特征无关。     

Mutations responsible for Mycobacterium tuberculosis isoniazid resistance in Italy.

SETTING: The incidence of tuberculosis (TB) and drug resistance in Italy is low compared to other countries. Mutations in several genomic regions of Mycobacterium tuberculosis are involved in the occurrence of isoniazid (INH) resistance. OBJECTIVE: To investigate the mutations responsible for INH resistance among Italian isolates of M. tuberculosis, to assess the feasibility of predicting drug resistance using a genetic approach. DESIGN: The mutations responsible for INH resistance were looked for in selected regions of genes katG, kasA and ndh and in the promoter regions of inhA and ahpC by nucleotide sequencing, and the results were compared with data reported in other studies. RESULTS: Prevalent INH resistance mutations were found at codon 315 of the katG gene and at position -15 of the inhA regulatory region (respectively 37.8% and 20.0% of isolates). The prevalence of mutations at position -24 of inhA, in ahpC, and in kasA ranged from 2.2% to 4.4%. No mutations were found in 35.6% of the isolates. CONCLUSION: The identification of INH resistance by genetic analysis of the selected regions may be inappropriate in areas with a low prevalence of TB, such as Italy, as the genetic mechanisms of resistance remain unidentified for approximately one third of the isolates.     

重庆地区结核分枝杆菌异烟肼和丙硫异烟胺耐药及其交叉耐药相关基因突变研究

目的 研究结核分枝杆菌(MTB)对异烟肼(INH)和丙硫异烟胺(Pto)的耐药情况,分析INH耐药相关基因(katG、inhA、ahpC、kasA)及与Pto交叉耐药相关基因的突变特点。方法 对233株INH耐药MTB临床分离株(37株对Pto同时耐药)的katG、inhA、ahpC、kasA基因进行扩增及序列分析。结果 耐异烟肼菌株中未发现katG完全缺失,223株 (96.5%)耐药株katG存在点突变、插入,其中 2个突变位点未见报道。195株 (83.6%)耐药株的第 315位点突变;189株 (81.1%)耐药株第463位点突变(R463L),其中166株 (71.2%)与第 315位点联合突变;在其他位点发生突变的菌株4株,包括D448G 1株、D419H 1株和2株同义突变。 11株(4.72%) INH耐药菌株中inhA点突变,包括S94A 1株、I194T 1株,C-15T 9株。37株Pto耐药的菌株中,8株点突变且均为inhA C-15T(4株为inhA单基因点突变C-15T,4株发生katG S315T和inhA C-15T的双基因联合突变)。全部耐药菌中有1株为ahpC基因突变,未发现有kasA基因突变。结论 进一步证实katG315和inhA-15基因位点突变与MTB对INH耐药密切相关;INH与Pto存在着交叉耐药,但耐药率较低,Pto耐药的发生与inhA-15位点突变密切相关。     

多重聚合酶链反应-单链构象多态性分析检测耐异烟肼结核分枝杆菌

目的　建立耐异烟肼 (isoniazid ,INH )结核分枝杆菌多重聚合酶链反应 单链构象多态性分析 (multiple polymerasechainreaction singlestrandconformationpolymorphism ,multi PCR SSCP)方法 ,快速、特异地同时检出aphC启动子、inhA、katG基因的突变情况 ,用于快速诊断结核分枝杆菌对INH的耐药性。方法　根据结核分枝杆菌的aphC启动子序列、inhA序列、katG序列 ,分别设计出 3对特异性寡聚核苷酸引物 ,采用multi PCR及SSCP技术 ,同时检测对结核分枝杆菌耐INH起作用的这 3个基因的突变情况。结果　对H3 7Rv标准株、临床分离INH敏感株 (2 3株 )及INH耐药株 (3 5株 )分别采用常规PCR和multi PCR同时进行扩增 ,两种扩增方法均能扩增出预期的目的片段 ,且结果符合率达10 0 % ;采用单基因PCR SSCP ,aphC启动子序列突变检出率 17% (6/3 5)、inhA序列突变检出率 2 0 % (7/3 5)、katG序列突变检出率 66% (2 3 /3 5) ;multi PCR SSCP突变检出率 83 % (2 9/3 5)。结论　耐药基因检测指导治疗是一种新探索 ,multi PCR SSCP方法敏感、特异 ,能同时快速有效地检测结核分枝杆菌aphC启动子、inhA、katG 3个INH耐药基因的突变 ,提高检验效率 ,有望成为临床指导用药的好方法。     

徐州市耐多药结核耐药分析及分枝杆菌基因突变特征研究

目的 研究徐州市耐多药结核(MDR-TB)耐药表型、耐INH或RFP相关基因突变情况,分析耐药表型与基因突变间关系,为耐多药结核疾病的诊断提供科学依据。方法 采取随机方法抽取徐州市115例MDR-TB菌株和66株全敏感菌株进行耐药情况分析,使用基因芯片检测技术对耐INH相关基因katG、 inhA和aphC以及耐RFP相关基因rpoB突变位点进行检测,对结果进行t检验分析。结果 徐州市MDR-TB菌株耐药表型有9种组合,主要是以耐INH+RFP组合为主,比例为47.83%,其次为耐INH+RFP+SM组合,比例为20.00%。与耐INH的相关基因突变率87.83%,基因突变类型分别为单基因katG(64.35%)和inhA(3.48%),双基因katG+inhA(12.17%)和katG+aphC(12.17%)。耐多药株(115例)和敏感株(66例)总体变异率均数比较(t=107.56,P<0.05), 其中katG基因突变率比较(P<0.05),二者差异均有统计学意义。与耐RFP相关rpoB基因总突变率86.09%,耐RFP相关rpoB基因突变类型分别为单531(45.22%)、516(8.70%) 和526位点,双(531+516)(13.91%)、(531+513)(12.17%)和516+533突变位点, 531+516+513(3.48%)三位点突变。耐多药株和敏感株总体和单个位点突变率均数比较(t=94.92,P<0.05), 531(P<0.05)、516(P<0.05)、533(P<0.05),二者差异均有统计学意义。结论 研究发现了徐州市MDR-TB耐药表型特征。徐州市MDR-TB菌株与耐INH和RFP相关基因突变客观存在,并表现出多态性和地区性。与耐INH相关katG基因、与耐RFP的相关rpoB基因531、516、533位点突变相对稳定,有临床应用价值,可作为徐州市耐药结核菌株快速诊断的指标。     

基因芯片快速检测结核分枝杆菌katG基因突变及其与异烟肼耐药相关性

目的了解结核分枝杆菌katG基因S315突变与异烟肼（isoniazid,INH）耐药相关性,建立快速简便的katG基因S315突变基因芯片检测方法。方法采用二倍稀释法检测123株结核分枝杆菌临床分离菌株对INH的耐药性。PCR及测序确定上述结核分枝杆菌katG基因S315位点突变率及突变类型。根据PCR及测序结果,设计Cy3荧光标记探针并制备katG基因S315位点突变检测基因芯片。基因芯片检测结果与PCR及测序结果进行对比分析。结果 123株结核分枝杆菌临床菌株中,39.0%（48/123）菌株对INH敏感,61.0%（75/123）菌株对INH耐药。结核分枝杆菌H37Rv株及所有临床菌株均能扩增出katG基因片段。75株INH耐药菌株中,69.3%（52/75）菌株katG基因出现S315位突变,其中25株突变类型为S315N（AGC→AAC）、12株为S315T（AGC→ACC）、7株为S315I（AGC→ATC）、各有3株分别为S315T（AGC→CGC）和S315R（AGC→AGA）、2株为S315G（AGC→GGC）。所制备的基因芯片对123株结核分枝杆菌katG基因S315野生型或突变型检测结果与PCR及测序结果完全一致。结论结核分枝杆菌katG基因S315突变与INH耐药密切相关。本研究中制备的基因芯片可快速、简便、准确、敏感和特异地检测结核分枝杆菌katG基因S315突变及其类型。     

Application of molecular methods for detection and transmission analysis of mycobacterium tuberculosis drug resistance in patients attending a reference hospital in Italy

A molecular analysis of drug-resistant isolates of Mycobacterium tuberculosis was done in a population with a high prevalence of human immunodeficiency virus infection. Seventy-one consecutive isolates were tested for genotypic resistance to isoniazid, rifampicin, streptomycin, and ethambutol by polymerase chain reaction-single strand conformation polymorphism analysis and automated sequencing of target regions. Phenotypic and genotypic resistance to isoniazid, rifampicin, streptomycin, and ethambutol were detected in 23.4%, 11.2%, 7%, and 5.6% of isolates and in 87%, 88%, 40%, and 100% of resistant isolates, respectively. Specificity was 100% for all target regions. When rpoB, katG, and ahpC mutation analysis were combined, 86% of resistant isolates to any drug were identified. No mutations in inhA were found in isoniazid-resistant isolates. Molecular detection of drug resistance, particularly for isoniazid and rifampicin, may represent a sensitive and very specific technique. The strategy of selecting rpoB, katG, and ahpC to quickly identify most resistant isolates, with a relevant saving of resources, is warranted.     

Genotypic analysis of isoniazid and rifampin resistance in drug-resistant clinical Mycobacterium tuberculosis complex isolates in southern Turkey

In this study we aimed to learn about the nature and frequency of katG, inhA, and rpoB gene mutations underlying isoniazid (INH) and rifampin (RMP) resistance in clinical Mycobacterium tuberculosis complex isolates. The Silver Sequence DNA sequencing method was used to detect the resistance condition of 22 INH, 6 RMP, and 13 INH and RMP in previously determined drug-resistant clinical M. tuberculosis isolates. Thirty of 35 (85.7%) INH-resistant strains and 14 of 19 (73%) RMP-resistant strains were found to have a mutation in the analyzed katG gene fragment or inhA locus and rpoB gene fragment. In the katG gene region, the codons of mutation detected were determined to be 315 (23 of 30, 76.6%), 279 (4 of 30, 13.3%) and 293 (1 of 30, 3.3%), a finding that has not been reported previously. Our findings demonstrated that the most frequent mutation pattern was Ser315Thr at codon 315 with a rate of 60% (18 of 30). In 5 (16.6%) isolates, a nucleotide change was detected which is associated with INH resistance from -15(th) C to T in the inhA locus. In the rpoB gene region, codons possesing point mutations were 531 (9 of 14, 64.2%), 516 (1 of 14, 7.1%), 524 (1 of 14, 7.1%), and 545 (4 of 14, 28.6%), which has not been reported previously. We believe about that our present study supplies important data on the different kinds of mutations occurring at various target loci for associated RMP and INH resistance in clinical isolates of our restricted region.     

Molecular characterization of drug-resistant mycobacterium tuberculosis isolates from Sichuan Province in china

Tuberculosis is still a severe public health issue in eastern Asia, and Sichuan is the key area for tuberculosis control in China. To determine the phenotypic and mutation patterns of drug resistance in Mycobacterium tuberculosis isolates from Sichuan, the drug susceptibility of 198 clinical isolates was examined. Among these isolates, 76 drug-resistant and 20 susceptible isolates were analyzed for the rpoB, embB, and katG and inhA regulatory regions. These are mutations believed to associate with rifampin (RIF), ethambutol (EMB), and isoniazid (INH) resistance, respectively. Of the 60 RIF-resistant isolates, 54 (90.0%) carried mutations on the amplified fragment of the rpoB gene, and the most common one (64.8%, 35/54) was at codon 531. Two new mutation patterns were recognized: one isolate harbored three mutations at codons 511, 516, and 518, and the other carried the dual mutation GAChACC at codon 516. A total of 30 INH-resistant isolates (60.0%, 30/50) had mutations at codon 315, whereas 4 (8.0%) had mutations at the inhA regulatory region. Among the 46 EMB-resistant isolates, 22 harbored the Met306 mutation. The results showed geographical variation in the mutation types of drug-resistant genes in M. tuberculosis isolates from Sichuan; this finding is valuable for the development of targeted and rapid molecular diagnostic methods suitable for specific regions.     

基因芯片技术检测贵州省结核分枝杆菌耐药基因KatG和inhA

目的 了解本地区结核分枝杆菌(MTB)异烟肼(INH)相关耐药基因katG和inhA的突变特征,评价基因芯片法对MTB INH耐药性检测的临床应用价值。方法 应用基因芯片法对经PCR-荧光探针法鉴定为MTB阳性的2 738例病人标本进行INH耐药性检测,分析其相关耐药基因katG和inhA的突变特征,同时应用比例法检测上述同期送检的相同病人的同类型标本经罗氏培养MTB阳性菌株的INH耐药性,比较两种方法的检测结果。结果 2 738例MTB核酸阳性标本经基因芯片法检测,INH耐药465例,耐药率16.98%,其中以katG 315(AGC→ACC)突变为主,基因突变率为78.06%,其次为inhA -15(C→T),katG 315(AGC→AAC),突变率分别为16.13%和5.59%。上述病人同期送检的同类型标本有1 493例经罗氏培养MTB阳性,比例法检测,INH耐药255例,耐药率17.08%,对应的基因芯片法检测结果为INH耐药249例,耐药率16.68%。以比例法结果为判断标准,基因芯片法测定INH耐药性的敏感性、特异性、阳性预测值(PPV)、阴性预测值(NPV)及准确性分别为85.49%、97.50%、87.55%、97.03%和95.45%。结论 本地区INH耐药以katG 315(AGC→ACC)和inhA -15(C→T)突变类型为主;基因芯片对MTB INH耐药性检测具有高敏感性、特异性、准确性和快速性,可用于本地区MTB INH耐药性的快速检测。     

ahpC基因突变与结核分支杆菌耐异烟肼的研究

目的了解我国结核分支杆菌耐异烟肼（ＩＮＨ）分离株ａｈｐＣ编码基因及其启动子突变情况，研究其与ＩＮＨ耐药关系。方法通过聚合酶链反应（ＰＣＲ）单链构象多态性（ＳＳＣＰ）方法分析６２株结核分支杆菌临床分离株ａｈｐＣ及其启动子基因。以Ｈ３７ＲＶ标准株为对照。结果３２株结核分支杆菌药物敏感株ａｈｐＣ及其启动子基因ＳＳＣＰ均泳动正常；３０株耐ＩＮＨ分离株ａｈｐＣ编码基因ＳＳＣＰ也未见异常；１２株高度耐ＩＮＨ分离株中，５株ａｈｐＣ启动子ＳＳＣＰ泳动异常；１８株低度耐ＩＮＨ分离株的ａｈｐＣ启动子泳动正常。结论结核分支杆菌ａｈｐＣ编码基因与耐ＩＮＨ无关；ａｈｐＣ启动子突变是结核分支杆菌ｋａｔＧ改变、过氧化氢酶缺乏的代偿性变化，也许能作为结核分支杆菌高度耐ＩＮＨ的间接指标。     

MTBDR plus方法快速检测北京地区临床结核分枝杆菌分离株利福平和异烟肼耐药性效果评价

目的评估MTBDR plus试剂盒在北京地区快速检测利福平和异烟肼的耐药性的效果。方法筛选169例临床结核分枝杆菌分离株,以国际标准的比例法作为对照,探讨该试剂盒在临床上应用的敏感性和特异性以及突变分布频率。结果在北京地区使用MTBDR plus检测利福平和异烟肼的敏感性和特异性分别为96.9%、85.3%和93.2%、98.1%。rpoB基因频率最高的突变位点是S531L(55.2%),其次是D516V(8.3%)、H526Y(7.3%)和H526D(3.1%)。katG基因频率最高的突变位点是S315T1(62.9%),而inhA是C15T位点(21.6%)。结论 MTBDR plus是一个敏感、特异和快速的诊断利福平、异烟肼耐药性和MDR的有效方法。     

结核分支杆菌五种耐药基因检测的临床应用及评价

目的　检测结核分支杆菌rpoBkatG、rpsL、pncA和embB耐药基因 ,评价其临床应用价值。方法　采用聚合酶链反应 单链构象多态性 (PCR SSCP)分析和药敏试验 (比例法 ) ,了解 10 9例肺结核患者结核分支杆菌耐药情况 ,并分析、比较临床治疗效果。结果　 1/ 2以上的肺结核患者至少耐两种抗结核药物 ,对RFP、INH、SM、PZA和EB总耐药率分别为 80 7%、71 5 %、78 8%、5 7 7%和48 6%。rpoB、katG、rpsL、pncA和embB基因突变率分别为 76%、68%、71%、5 1%和 3 0 %。结核分支杆菌耐药基因突变率与耐药水平联系密切 ,多数结核分支杆菌耐药基因突变易发生在高耐药株 ,也有少数基因突变发生在低耐药株。根据药敏试验和耐药基因检测结果 ,6个月耐多药结核治愈率分别达到 5 4 8%和 62 8% ,治疗效果满意 ,两种方法差异无显著性 (P >0 0 5 )。结论　耐药基因检测指导治疗是一种新探索 ,PCR SSCP方法敏感、特异 ,可以快速检测结核分支杆菌rpoB、katG、rpsL、pncA和embB耐药基因突变 ,可能会成为临床指导用药的好方法     

结核分支杆菌inhA基因突变的测序研究

目的 对耐药结核分支杆的inhA基因进行测序分析以研究其变异的分子机制。方法 以inhA片段为引物,聚合酶链反应扩增H27Rv、耐药株和敏感株的inhA基因,产物克隆后制备质粒,ABI377全自动核酸测序系统测定DNA序列。结果 在17个耐异烟肼菌株中,11株出现inhA基因变异,突变率为65％,其中在10株至少每株有1个错义突变。变异主要为点突变,包括碱基置换和缺失。各菌株变异不同。katG和inhA同时变异的达62％。首次发现了18个与结核分支杆菌耐INH和EMB特性有关的inhA基因位点。结论 结核分支杆菌耐INH和EMB特性与inhA突变异致药物结合位点减少有关。     

GenoType MTBDRplus快速检测耐多药结核分枝杆菌复合群临床分离株效果评价

目的评价GenoType MTBDRplus快速检测耐多药结核分枝杆菌复合群(multi-drug resistant Mycobacterium tuberculosis Complex,MDR-MTBC)临床分离株的应用效能。方法随机抽取福建省30个耐药监测点的129株MTBC包括全敏感菌株50株和79株MDR菌株,应用MTBDRplus 检测,同时扩增MDR菌株的rpoB, katG, inhA基因进行测序比对,并将检测结果与比例法药敏结果比较分析。结果129株MTBC经MTBDRplus检测,Tub条带均阳性(100%),50株全敏感株的检测结果与比例法药敏结果完全一致。79株MDR菌株经MTBDRplus检出67株(84.81%)MDR,10株(12.66%)单耐RFP,2株(2.53%)单耐INH。经测序,77株MDR菌株的rpoB基因检测出突变,71株的katG 和/或inhA检测出突变。以比例法药敏结果为金标准,MTBDRplus检测RFP、INH耐药性及MDR的敏感度分别为97.47%(77/79), 87.34%(69/79), 84.81%(67/79),MTBDRplus检测RFP、INH耐药性及MDR的特异度均为100%(50/50),一致率分别为98.45%(127/129),92.25%(119/129),90.70%(117/129)。结论MTBDRplus是一种快速、准确、操作简便的技术,具有很好的应用前景,适用于耐多药结核分枝杆菌临床分离株的快速检测,但灵敏度有待进一步提高。     

Mtb耐异烟肼基因变异株的体外最小抑菌浓度研究

目的 研究Mtb耐INH临床分离株katG基因、inhA基因、ahpC基因突变对其体外最小抑菌浓度（MIC）的影响。方法 对临床分离的耐INH的160株Mtb及对INH敏感的40株Mtb进行耐药基因芯片检测;并对所有标本进行INH MIC测定,比较不同变异株的MIC结果,其数据采用t检验进行比较,以P＜0.05为差异有统计学意义。结果 Mtb耐INH株katG 315基因变异占68.8%（110/160）,katG 315基因变异株的MIC为（20.20±19.46 μg/ml）,高于inhA变异株（0.73±0.82 μg/ml）（t′=10.9171,t′_α=1.9807,P＜0.05）;低于katG 315+inhA变异株（69.33±32.45 μg/ml）,（t′= 7.164,t′_α= 2.0627,P＜0.05）。结论 Mtb耐INH基因有不同的突变模式,各模式存在不同的耐药程度,此可以给临床用药进一步提供参考。     

Rifampin and isoniazid resistance associated mutations in Mycobacterium tuberculosis clinical isolates in Seville, Spain.

The susceptibility phenotypes of 964 clinical isolates of Mycobacterium tuberculosis were studied over a 7-year period in Seville, Spain. Thirty-eight (3.9%) strains were rifampin (RMP) resistant, 79 (8.2%) were isoniazid (INH) resistant and 22 (2.3%) were resistant to at least both antimicrobials (multidrug-resistant, MDR). We studied the mechanisms of resistance to these drugs in 94 resistant clinical isolates of M. tuberculosis using three molecular methods: 1) PCR-single strand conformation polymorphism (SSCP) analysis, 2) RFLP analysis using B1/B2 primers, and 3) sequence analysis. Five different mutations were detected in the rpoB gene: Ser531-->Leu (72.3%), His526-->Asp (12.8%), Asn518-->Ser (2.1%), Gln513-->Leu (2.1%) and a nine-nucleotide deletion (2.1%). In the case of resistance to INH, four different mutations in the katG gene were detected, Ser315-->Thr (58.0%), Ser315-->Leu (2.9%), partial deletion (5.8%) and Ile304-->Val (1.4%), while in the inhA regulatory region the only mutation was the nucleotide substitution C209T (4.3%). No mutation was found in the ahpC promoter.     

应用基因芯片方法检测结核分枝杆菌利福平和异烟肼的耐药性

目的 应用基因芯片方法检测结核分枝杆菌(Mycobacterium tuberculosis,Mtb)对利福平和异烟肼的耐受性,评价其临床应用价值。方法 应用聚合酶链反应（polymerase chain reaction,PCR）扩增-基因芯片杂交的方法检测经常规药敏实验证实的30株Mtb利福平和异烟肼敏感株和50株耐利福平和异烟肼分离株的rpoB基因及katG和inhA基因突变,同时以PCR-直接测序法为对照。结果 应用PCR-基因芯片与基因测序方法检测30株Mtb利福平敏感株rpoB基因和异烟肼敏感株katG基因和inhA基因均为野生型。50株Mtb利福平耐药株中,PCR-基因芯片与基因测序分析3株rpoB基因均为野生型,41株均为突变型;6株PCR-基因芯片与基因测序结果不一致。50株Mtb异烟肼耐药株中,PCR-基因芯片与基因测序分析16株katG基因和30株inhA基因均为野生型,31株katG基因均为315位密码子突变,7株inhA基因均为15位突变型,其中2株为katG和inhA双重突变;3株katG和13株inhA PCR-基因芯片与基因测序结果不一致。结论 应用PCR-基因芯片方法可快速、有效地检出大多数Mtb耐多药分离株,指导临床用药。