快速检测结核分支杆菌对异烟肼的耐药性的临床应用

目的建立快速检测结核分支杆菌耐药基因的分子药敏方法。方法要聚合酶链反应——单链构象多态性（PCR—SSCP）分析40株结核分支杆菌KatG基因突变。其中20株为异烟肼（INH）敏感株,20株为INH耐药株,用PCR—SSCP图谱鉴定扩增产物有无突变,H37RV标准株作对照．结果所有INH敏感株SSCP带普与对照相同;19株INH耐药株中8株与对照相同,11株有不同程度的差异,INH耐存KatG基因突变或缺失的阴性率为60％。结论多数结核分支杆菌INH是由于其KatGj基因突变所致,用PCR—SSCP筛选突变株可达到快速检测结核分支杆菌INH耐药的目的。     

PCR-DNA测序技术检测结核分支杆菌耐多药基因突变的研究

目的应用PCR—DNA测序技术快速检测同时耐利福平和异烟肼结核分支杆菌分离株rpoB、KatG基因突变，评价其在检测结核分支杆菌耐多药性方面的价值。方法47株耐利福平和异烟肼结核分支杆菌临床分离株及30株结核分支杆菌敏感分离株用PCR—DNA测序技术分别检测rpoB、KatG基因突变。结果47株耐多药结核分支杆菌分离株中，检出43株rpoB基因突变，突变检出率为91．5％（43／47）；31株KatG基因突变，突变检出率为66．0％（31／47）；rpoB和KatG基因同时突变者31株，突变检出率为66．0％（31／47）。30株结核分支杆菌敏感株检出1株KatG基因突变。结论PCR—DNA测序技术方法敏感、准确、特异，可快速检测结核分支杆菌rpoB、KatG耐药基因突变，有利于耐多药结核分支杆菌耐药性的快速检测。     

结核杆菌异烟肼表型耐药与katG基因突变相关性研究

目的:探讨结核分支杆菌异烟肼(INH)表型耐药与KatG基因密码子315位和463位基因改变的相关性。方法:采用基因直接测序法对结核分支杆菌分离株KatG基因进行分析。结果:132株异烟肼耐药菌株中68株KatG基因密码子315位有基因突变,突变率51.52%(68/132),野生型密码子315AGC(Ser)突变成ACC(Thr)60株,AAC(Asn)6株,ACA(Thr)1株,GGC(GIy)1株。133株INH敏感菌株未发现KatG 315位基因突变。132株INH耐药菌株中123株KatG基因密码子463位有基因突变,突变率为93.18%,野生型密码子463CGG(Arg)突变成CTG(Leu)123株;133株敏感菌株中114株KatG基因密码子463位有基因突变,突变率为85.71%,野生型密码子463 CGG(Arg)突变成CTG(Leu)114株。结论:①KatG基因315位密码子突变是菌株异烟肼表型耐药的重要分子基础,以Ser315Thr错义突变为主。②KatG基因463位密码子突变与异烟肼耐药表型没有相关性,不是结核杆菌异烟肼耐药的分子标志。该位点突变可能是基因多态性,与结核菌治疗带来的选择性压力无关。     

中国部分地区结核分支杆菌耐利福平、异烟肼和链酶素耐药基因的检测

目的研究我国部分地区耐利福平（RFP）、异烟肼（硼）和链酶素（SM）结核分支杆菌临床分离株rpoB基因、KatG、rpsL基因突变情况，评价其耐药分子机制。方法采用PCR和聚合酶链反应．单链构象多态性（PCR-SSCP）对373株结核分支杆菌临床分离株（其中敏感株148株，耐RFP、INH、SM株共225株）rpoB基因、KatG和砒基因进行检测。并对其中19株SSCP阴性的耐RFP株用双脱氧末端终止法进行测序分析。结果安徽省、北京市、上海市、河北省、河南省、辽宁省、吉林省结核分支杆菌耐RFP临床分离株rpoB基因突变率分别为90％、91％、90％、91％、90％、93％、91％；KatG基因突变率分别为69％、63％、71％、68％、67％、68％、65％，rpsL基因突变率71％、69％、74％、68％、70％、61％、76％，分别为经统计学处理不同地区间无显著性差异（X^2=0．46，P＞0．05）。同时rpoB基因敏感株均无突变，特异性为100％；KatG基因有3株发生突变，特异性为98％。19株SSCP阴性耐药株中经测序6株在531位密码子TCG→TIE，1株526位密码子CAC→TAC，7株发生在516位密码子GAC→GTC，5株未改变。结论我国不同地区耐利福平、异烟肼和链霉素的rpoB、KatG、rpsL耐药基因突变率大致相同，rpoB基因、KatG和rpsL基因突变分别是耐RFP、INH和SM结核分支杆菌耐药性产生的主要分子机制，PCR-SSCP可快速检测结核分支杆菌RFP、INH和SM耐药性。     

结核杆菌耐药性调查与基因突变分析

目的了解结核分支杆菌耐药性与基因突变情况,分析耐药表型与基因突变的相关性。方法从临床标本中分离培养、鉴定结核分支杆菌;应用药敏试验、单链探针反向杂交试验技术(LiPA)和基因测序分析结核分支杆菌耐药性与基因突变情况。结果50株结核分支杆菌中,12株耐异烟肼(INH),7株耐利福平(RFP),15株耐链霉素(SM),2株耐乙胺丁醇(EMB)。LiPA检测耐RFP菌株,4株rpoB基因531位TCG→TTG突变;耐INH菌株中5株KatG基因315位AGC→ACC突变;耐SM菌株中6株、SM敏感株1株rpsl基因43位AAG→AGG突变,1株耐SM菌株rpsl基因88位AAG→AGG突变;rrs基因未检测到突变;1株耐EMB菌株与1株EMB敏感株的embB基因306位发生ATG→GTG突变。结论本市结核分支杆菌的耐药性增加的趋势显著,加强监测与综合干预非常必要。     

PCR-SSCP方法用于痰标本中结核分支杆菌耐药基因的检测

目的探讨采用直接检测结核病人痰标本中rpoB基因和KatG基因突变。评价该方法检测结核分支杆菌对利福平（RFP）和异烟肼（INH）耐药性。方法用PCR-SSCP技术分析，对35例耐INH（或含耐异烟肼）、63例耐RFP（或含耐RFP）的肺结核病人痰标本和20例非结核性肺部疾病组痰标本进行rpoB和KatG基因突变的检测。结果以结核分支杆菌H37RV为对照，63例耐RFP痰标本中57例PCR扩增阳性，其中46例SSCP图谱与H37Rv标准株有差异。rpoB突变率为65．2％。35例耐INH肺结核病人痰标本有20例扩增阳性。15例SSCP图谱与H37Rv标准株有差异，突变率为42．8％。20例非结核病人痰标本rpoB基因和KatG基因扩增均为阴性。结论PCR-SSCP技术有望成为结核分支杆菌耐药性检测的重要方法之一。     

聚合酶链反应-测序技术快速检测耐异烟肼结核分枝杆菌KatG基因突变的研究

目的应用PCR-DNA测序技术快速检测耐异烟肼结核分枝杆菌分离株KatG基因突变,评价其在检测结核分枝杆菌异烟肼耐药性方面的应用价值。方法47株耐异烟肼结核分枝杆菌临床分离株及30株结核分枝杆菌敏感分离株用PCR-DNA测序技术检测KatG基因突变。结果47株耐异烟肼结核分枝杆菌分离株中,有31株KatG基因检出有突变,突变检出率为66.0%(31/47);30株结核分枝杆菌敏感株检出1株KatG基因突变。结论PCR-DNA测序技术方法敏感、准确、特异,可快速检测结核分枝杆菌KatG耐药基因突变,有利于耐异烟肼结核分枝杆菌耐药性的快速检测。     

聚合酶链反应-测序技术快速检测耐异烟肼结核分枝杆菌KatG基因突变的研究

目的 应用PCR-DNA测序技术快速检测耐异烟肼结核分枝杆菌分离株KatG基因突变,评价其在检测结核分枝杆菌异烟肼耐药性方面的应用价值.方法 47株耐异烟肼结核分枝杆菌临床分离株及30株结核分枝杆菌敏感分离株用PCR-DNA测序技术检测KatG基因突变.结果 47株耐异烟肼结核分枝杆菌分离株中,有31株KatG基因检出有突变,突变检出率为66.0%(31/47);30株结核分枝杆菌敏感株检出1株KatG基因突变.结论 PCR-DNA测序技术方法敏感、准确、特异,可快速检测结核分枝杆菌KatG耐药基因突变,有利于耐异烟肼结核分枝杆菌耐药性的快速检测.     

结核分支杆菌rpoB基因突变与对利福平耐药的关系

目的:研究结核分支杆菌耐利福平(RFP)分离株rpoB基因突变情况并评价英应用价值.方法:采用多聚合酶链式反应-单链构象多态性分析法(PCR-SSCP法)对60株结核分支杆菌rpoB基因进行检测.结果:以结核分支杆菌标准株H37Rv为对照,60株PCR扩增阳性的结核分支杆菌中,共有32株发生突变,总突变率为53.3%(32/60);24株药物敏感株有4株rpoB基因有突变,突变率为16.7%;36株耐药株中,28株rpoB基因有突变,突变率为77.8%.耐药菌株突变率明显高于敏感菌株的突变率(P<0.05).结论:rpoB基因突变是耐RFP结核分支杆菌耐药性的主要分子机制;应用PCR-SSCP可快速检测结核分支杆菌对RFP的耐药性.     

PCR-SSCP法检测耐多药结核分支杆菌耐药性

我国是结核病疫情最严重的国家之一，其主要原因是耐药和耐多药结核病的广泛分布和迅速传播。异烟肼（INH）和利福平（RFP）均是抗结核的一线药物，对于其作用机理及结核杆菌的耐药机制的研究日益受到重视。据报道认为结核分支杆菌耐与RNA聚合酶的8亚基的编码基因rpoB的突变有关；耐异烟肼的产生是过氧化氢酶一过氧化物酶的编码基因KatG基因的缺失和突变有关，也inhA基因突变也有相关性。我们采用PCR—SSCP银染法直接检测临床分离株中结核分支杆菌rpoB基因和KatG基因突变，现将结果报告如下。     

结核分枝杆菌katG、inhA、ahpC等突变与异烟肼耐药关系的研究

目的：了解结核分枝杆菌katG、inhA、ahpC、fabG1、sodA及sodC基因突变的特征及其与耐异烟肼的关系。方法对127例活动性肺结核患者痰标本进行菌型鉴定及结核分枝杆菌药敏试验,提取结核分枝杆菌菌株DNA,应用PCR扩增katG、inhA及ahpC、fabG1、sodA及sodC基因片段,并进行DNA序列分析。结果结核分枝杆菌药物敏感试验显示127株结核分枝杆菌中,其中47株耐异烟肼,80株对异烟肼敏感,耐异烟肼率为37.01%。47株耐异烟肼中,29株存在katG和（或）inhA基因突变,其中22株（46.81%,22/47）存在katG基因单位点突变,3株（6.38%,3/47）存在inhA基因单位点突变,4株（8.51%,4/47）存在katG及inhA基因联合位点突变。22株katG基因单位点突变中,20株为AGC315ACC、AGC315AAC （42.55%,20/47）突变,2株（2.13%,1/47）分别为CTG378CCG（Leu378Pro）、ACG394ATG（Thr394Met）突变,该突变位点及突变形式尚未见文献报道。18株katG及inhA未突变结核分枝杆菌均未检测到ahpC、fabG1、sodA及sodC基因突变。结论结核分枝杆菌对异烟肼耐药主要与katG和inhA基因突变有关。耐异烟肼结核分枝杆菌临床分离株378和394新突变位点的发现为进一步研究耐药机制以及耐药结核病的快速检测提供了依据。     

应用多重PCR-单链构象多态性分析同时检测耐异烟肼和利福平的结核分枝杆菌

目的 探讨应用多重PCR-单链构象多态性分析(multiplexpulymerase chain reaction-single strand conformation polymorphism,multi-PCR-SSCP)方法快速、特异地同时快速检测结核分枝杆菌对异烟肼和利福平耐药性的效能.方法 根据结核分枝杆菌的inhA序列、katG序列、rpoB序列,分别设计出3对特异性寡聚核苷酸引物.采用multi-PCR-SSCP技术,一次性检出耐异烟肼和利福平的结核分枝杆菌.新方法的有效性通过116株临床分离株(70株耐异烟肼,66株耐利福平)的验证.结果 名 Multi-PCR-SSCP方法检测临床分离株基因突变的有效性,以细菌培养和药敏试验结果为金标准.116株临床分离株和H37Rv标准株中除了4株katG缺失突变,其余菌株3个基因katG、inhA和rpoB在单基因PCR中都扩增成功.与H37Rv标准株相比,46株katG基因突变,14株inhA基因突变,58株rpoB基因突变.38株katG和rpoB,4株inhA和rpoB,4株inhA和katG同时突变,还有2株3个基因都有突变.multi-PCR-SSCP对于耐异烟肼和利福平的结核分枝杆菌检出的敏感度分别为80%、82%,特异度分别为100%和92%.结论 multi-PCR-SSCP方法敏感、特异,能同时快速有效地检测耐多药结核分枝杆菌,有望成为临床指导用药的好方法,为深入研究耐药基凶检测奠定了良好的基础.     

Mutations in katG gene sequences in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis are rare.

In this study, a battery of oligonucleotides was directed toward the katG gene and PCR-single-stranded conformation polymorphism (SSCP) analysis was used to search for katG gene deviations in clinical isolates of Mycobacterium tuberculosis from different geographical regions. Since a complete deletion of the katG gene was not found, it is suggested that deletion is not a major mechanism of isoniazid (isonicotinic acid hydrazide; INH) resistance in these isolates. However, 7 of 39 isolates (4 of 25 from South Africa and 3 of 14 from other geographical regions) showed mobility shifts by SSCP analysis, suggesting aberrations in the katG gene. Direct sequence analysis confirmed that the mobility shifts were due to Thr-275-->Ala (Thr275Ala), Arg409Ala, Arg463Leu, and Asp695Ala mutations and a 12-bp deletion in the 5' region of the katG gene. Mutations at codons 275, 463, and 695 created altered restriction sites for HhaI, MspI, and HaeIII, respectively, and sequence results, supported by restriction fragment length polymorphism analysis, suggested that the PCR-SSCP procedure is a good indicator of mutations in PCR-amplified fragments. Identical mutations at codons 463 and 275 were found in isolates from different geographical regions. This may suggest a common evolutionary event, but one of the control isolates (susceptible to INH [3%; n = 30]) also had a mutation at codon 463. The results suggest that variations in the katG coding gene sequences of INH-resistant isolates of M. tuberculosis are infrequent and that defects in other regions of the M. tuberculosis genome are of equal or greater importance in contributing to the acquisition of resistance to INH.     

Characterization of the katG and inhA genes of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

Resistance to isoniazid in Mycobacterium tuberculosis has been associated with mutations in genes encoding the mycobacterial catalase-peroxidase (katG) and the InhA protein (inhA). Among the 26 isoniazid-resistant clinical isolates evaluated in this study, mutations in putative inhA regulatory sequences were identified in 2 catalase-positive isolates, katG gene alterations were detected in 20 strains, and 4 isolates had wild-type katG and inhA genes. Mutations in the katG gene were detected in all 11 catalase-negative isolates: one frameshift insertion, two partial gene deletions, and nine different missense mutations were identified. An arginine-to-leucine substitution at position 463 was detected in nine catalase-positive isolates. However, site-directed mutagenesis experiments demonstrated that the presence of a leucine at codon 463 did not alter the activity of the M. tuberculosis catalase-peroxidase and did not affect the capacity of this enzyme to restore isoniazid susceptibility to isoniazid-resistant, KatG-defective Mycobacterium smegmatis BH1 cells. These studies further support the association between katG and inhA gene mutations and isoniazid resistance in M. tuberculosis, while also suggesting that other undefined mechanisms of isoniazid resistance exist.     

聚合酶链反应-单链构象多态性技术快速筛选结核杆菌的利福平耐药性

目的：探讨利用分子生物学方法直接快速检测结核分枝杆菌利福平（RFP）药物敏感性的可行性。方法：应用聚合酶链反应-单链构象多态性（PCR-SSCP）技术分别检测了40株RFP药物敏感及耐药的结核分枝杆菌临床分离株。先用PCR扩增rpoB基因，随后用SSCP方法鉴定其扩增产物有无突变，并与药敏试验结果作对照分析。结果：所有临床分离株均观察到rpoB基因PCR扩增产物。40株RFP敏感的临床分离株SSCP带谱与结核分枝杆菌H37RV标准株相同，40株RFP耐药株中37株检测到突变图谱。与Bactec结果对照，用PCR-SSCP技术检测结核分枝杆菌RFP耐药性的敏感性为93％，特异性为100％。结论：结核分枝杆菌耐RFP是其rpoB基因突变所致。PCR-SSCP技术可用于结核分枝杆菌RFP药物敏感性的直接快速检测。     

Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC -46A polymorphism

The present study investigated the prevalence and diagnostic potential of the most commonly reported mutations associated with isoniazid resistance, katG 315Thr, katG 315Asn, inhA -15T, inhA -8A, and the oxyR-ahpC intergenic region, in a population sample of 202 isoniazid-resistant Mycobacterium tuberculosis isolates and 176 randomly selected fully sensitive isolates from England and Wales identified by using a directed oligonucleotide array and limited DNA sequencing. The strains were recovered from patients originating from 29 countries; 41 isolates were multidrug resistant. Mutations affecting katG 315, the inhA promoter, and the oxyR-ahpC intergenic region were found in 62.7, 21.9, and 30% of 169 genotypically distinct isoniazid-resistant isolates, respectively, whereas they were found in 0, 0, and 8% of susceptible strains, respectively. The frequency of mutation at each locus was unrelated to the resistance profile or previous antituberculous drug therapy. The commonest mutation in the oxyR-ahpC intergenic region, ahpC -46A, was present in 23.7% of isoniazid-resistant isolates and 7.5% of susceptible isolates. This proved to be a phylogenetic marker for a subgroup of M. tuberculosis strains originating on the Indian subcontinent, which shared IS6110-based restriction fragment length polymorphism and spoligotype features with the Delhi strain and Central Asian strain CAS1; and this marker is strongly associated with isoniazid resistance and the katG 315Thr mutation. In total, 82.8% of unrelated isoniazid-resistant isolates could be identified by analysis of just two loci: katG 315 and the inhA promoter. Analysis of the oxyR-ahpC intergenic region, although phylogenetically interesting, does not contribute significantly to further identification of isoniazid-resistant isolates.     

Oxidative stress increases susceptibility of Mycobacterium tuberculosis to isoniazid

Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an approximately 200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.     

ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates

Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were > or =50 microg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were > or =100 microg/ml, and these isolates were also resistant to > or =8 microg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were > or =32 microg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to > or =50 microg of ETH per ml had such mutations.