深圳市耐多药结核分枝杆菌流行株耐药基因序列分析

目的 了解深圳市耐多药结核分枝杆菌（multidrug-resistantMycobacterium tuberculosis ,MDR-TB）的分子流行病学特征。 方法 参照WHO/IUATLD标准,使用L-J药敏培养基,采用1％比例法药敏试验筛选出敏感株和异烟肼（isoniazid,INH）、利福平（rifampicin, RFP）双耐药临床分离株,通过DNA测序检测深圳地区153株敏感株与132株MDR-TB的INH耐药基因 kat G、inh A、oxy R- ahp C基因间区域及RFP耐药基因 rpo B碱基排列顺序,运用DNASTAR和blastn进行序列分析。 结果 153株敏感株突变率为27.5%（42/153）。132株MDR-TB突变率为98.5%（130/132）,其中kat G基因突变率为73.5%（97/132）,68.9%（91/132）为 kat G315位突变; inh A基因突变率为18.2%（24/132）,11.4%（15/132）为启动子区域突变,未发现 inh A94特异突变株; ahp C基因突变率为16.7%（22/132）,10.6%（14/132）为启动子区域突变; rpo B基因81-bp核心区域突变率为93.2%（123/132）。 结论 kat G315、inh A启动子区域、ahp C启动子区域以及 rpo B 81-bp核心区域突变为深圳地区耐多药结核分枝杆菌主要突变类型,与其他国家和地区差异无统计学意义;但深圳地区未见 inh A94突变株。     

六安地区结核分枝杆菌耐药基因katG 和rpoB 的突变特征

目的 分析临床分离的结核分枝杆菌katG 和rpoB 基因的突变情况与耐药之间的关系,了解六安地区耐药结核分枝杆菌的基因突变特征。方法 采用比例法对六安地区65 株结核分枝杆菌临床分离株进行药敏试验;通过特异性引物,对目的基因片段katG 和rpoB 进行扩增、测序后进行分析。结果 60 株结核分枝杆菌中分别有有9 株耐异烟肼和14 株耐利福平,其中同时耐异烟肼和利福平的6株。9 株耐异烟肼菌株中,4 株katG 在315（Ａ GC→ACC 或ACA）位点发生突变,占44.4％;14 株耐利福平菌株中,11 株rpoB 分别在516（GAC→GTC）、526(CAC→CGC 或TAC)和531(TCG→TTG)位点发生突变,占总耐药菌株的78.6％;6 株同时对利福平和异烟肼耐药,其中5 株katG 或rpoB 基因发生突变,占83.3％。结论 六安地区结核分枝杆菌耐异烟肼和耐利福平分别主要是由katG 和rpoB 基因突变引起,其中耐异烟肼主要在315（AGC→ACC 或ACA）位,而耐利福平在516（GAC→GTC）、526(CAC→CGC或TAC)和531(TCG→TTG)位,都发生了突变。     

结核耐药基因rpoB/katG/inhA早期筛查MDR-TB的临床研究

目的评估结核耐药基因rpo B/kat G/inh A筛查耐多药结核的可行性及临床意义。方法对2016年1月至2017年5月我科住院的228例肺结核患者分别采用结核耐药基因检测法和改良罗氏培养比例法检测利福平、异烟肼是否耐药,比较两种检验方法诊断的一致性。结果以改良罗氏培养比例法为金标准,228例患者中结核耐药基因检测法利福平耐药的灵敏度为88. 24%,特异度为97. 63%,异烟肼耐药的灵敏度为92. 31%,特异度为99. 01%。两法有较高的一致性(无统计学差异)。结论结核耐药基因rpo B/kat G/inh A可早期、快速、准确检测利福平、异烟肼的耐药性,可作为耐多药结核早期筛查的方法,对结核病防与治有重要意义。     

利用DNA芯片技术检测新疆结核分枝杆菌rpoB基因与对利福平耐药水平的关系

目的研究新疆地区结核分枝杆菌rpoB基因的突变与对利福平耐药水平的关系。方法采用DNA芯片技术对耐利福平菌株rpoB基因进行检测。结果 46株结核分枝杆菌利福平初始耐药分离株,对利福平高耐药株30株,低耐药株13株。DNA芯片检测显示43株发生基因突变,低耐药株突变主要在526位点,少数在531位点;高耐药株主要在526位点及531位点上,其中3株为526和516位点同时发生联合突变,其余3株未见突变。结论以结核分枝杆菌标准株H37Rv为对照,新疆结核分枝杆菌对利福平耐药表现为结核分枝杆菌RNA聚合酶的β亚基的编码基因rpoB基因的点突变,而高耐药则表现为rpoB基因的526位和531位点突变及联合突变特征。     

结核分枝杆菌rpoB基因突变特征的初步探讨

目的 了解浙江省耐利福平结核分枝杆菌rpoB基因突变特征.方法 对65株临床分离菌株rpoB基因509-631位点分别进行PCR-SSCP检测和DNA序列测定,观察不同耐药株rpoB基因突变的规律.结果 耐利福平分离株96.4%（27/28）存在rpoB基因突变,其中526位突变率64.3%（18/28）,513位突变率21.4%（6/28）,531位突变率7.1%（2/28）,529位突变率3.6%（1/28）;耐其他抗结核药18.5%（5/27）存在rpoB基因突变,突变位点具有随机性.所有敏感菌株均无突变.结论 rpoB基因突变与利福平耐药密切相关,浙江省rpoB基因突变主要以526位突变为主,其次为513位,两者占总数的86%（24/28）,而耐其他抗结核药菌株也存在rpoB基因的突变,但没有明显规律,且均对利福平敏感.     

耐多药结核分枝杆菌基因突变分析

目的对临床分离的耐多药结核分枝杆菌株进行基因突变分析。方法对耐多药结核分枝杆菌临床分离菌株进行耐药基因的PCR检测,利用基因序列测定方法分析耐药基因突变情况。结果从耐多药结核病（MDR-TB）患者临床分离菌株中快速检测到rpoB、katG、rpsL、embB基因及其突变。耐多药菌株、全耐及单耐利福平分离菌株均检测rpoB基因点突变,突变位点主要为第516、526和531常见密码子,1例MDR-TB出现第479位和第531位密码子同时突变。耐多药菌、全耐菌及单耐异烟肼的菌株均检测有katG基因点突变,突变位点均为第2066位碱基C突变为Go全耐菌和对乙胺丁醇（EMB）耐药的耐多药菌株均检测有embB基因点突变,突变位点均为第306位密码子ATG突变为ACG。全耐菌和对链霉素（SM）耐药的MDR-TB菌株均检测有rpsL基因点突变,突变密码子为CCT突变为CTT,其中从1株对SM敏感的MDR-TB中也检测到突变。全敏感株、标准株及利福平（RIF）、异烟肼（INH）或EMB敏感的耐药株均无rpoB、katG或embB基因突变。结论PCR及基因序列测定可快速检测耐多药结核基因,结核分枝杆菌耐多药性与多个基因突变相关。     

结核分枝杆菌rpoB基因突变特征的初步探讨

目的了解浙江省耐利福平结核分枝杆菌rpoB基因突变特征。方法对65株临床分离菌株rpoB基因509-631位点分别进行PCR-SSCP检测和DNA序列测定,观察不同耐药株rpoB基因突变的规律。结果耐利福平分离株96.4%(27/28)存在rpoB基因突变,其中526位突变率64.3%(18/28),513位突变率21.4%(6/28),531位突变率7.1%(2/28),529位突变率3.6%(1/28);耐其他抗结核药18.5%(5/27)存在rpoB基因突变,突变位点具有随机性。所有敏感菌株均无突变。结论rpoB基因突变与利福平耐药密切相关,浙江省rpoB基因突变主要以526位突变为主,其次为513位,两者占总数的86%(24/28),而耐其他抗结核药菌株也存在rpoB基因的突变,但没有明显规律,且均对利福平敏感。     

应用基因芯片分析结核分枝杆菌常见耐药基因型的研究

目的应用基因芯片快速检测结核分枝杆菌耐药基因型,建立一种新的分子药敏试验方法。方法以传统药敏试验和PCR-直接测序方法为对照,应用基因芯片快速检测157株结核分枝杆菌临床分离株异烟肼(INH)、利福平(RFP)、链霉素(SM)和乙胺丁醇(EMB)耐药基因(katGr、poB、rp-sLr、rs和embB)的带荧光素标记的PCR产物。结果PCR-直接测序和基因芯片检测36株结核分枝杆菌药物敏感株的5种耐药基因均为野生型。121株结核分枝杆菌耐药分离株中,56株耐INH分离株,katG基因突变率为62.5%,其中katG缺失率为7.5%,315位密码子突变率为55.4%,279位密码子突变率为1.8%;104株耐RFP株,rpoB基因突变率为94.2%,最常见的突变位点为531位和526位密码子(突变率分别为60.6%、15.4%),双位点突变率为5.8%,还发现511、513、515、516、517、518和533位密码子突变;62株耐SM分离株,rpsL和rrs总突变率为88.7%(两者分别为82.3%、6.5%),rpsL突变位于43位和88位密码子(突变率分别为77.4%、4.8%),rrs突变位于513位和516位碱基(突变分别为4.8%、1.6%);57株为耐EMB株,embB基因突变率为61.4%,均为306位密码子突变,最常见的突变为ATG→GTG或ATA(突变率分别为35.1%、15.8%),还发现306位密码子ATG→ATC、ATT和CTG突变。通过基因芯片检出的突变与基因测序结果一致。结论应用基因芯片可分析大多数结核分枝杆菌耐药基因型,弥补传统药敏试验方法的不足,指导临床治疗。     

福建省龙岩市411例肺结核患者对利福平和异烟肼耐药状况的研究

目的 回顾性分析福建省龙岩市411例肺结核患者对利福平和异烟肼的耐药状况,为当地结核病防治工作提供科学依据。方法 收集2016年5月12日至2019年8月17日福建省龙岩市第二医院门诊及收住入院并确诊为肺结核的411例患者,其中初治患者312例,复治患者99例。作者对两组患者411株结核分枝杆菌临床分离株采用基因芯片技术进行利福平与异烟肼耐药性及其相关耐药基因突变位点的检测。采用Excel 2003和SPSS 20.0软件进行数据的整理和统计学分析,计数资料的比较采用χ ²检验,以P<0.05为差异有统计学意义。结果 411例肺结核患者中,初治患者利福平和异烟肼任一位点突变率[9.3%(29/312)]明显低于复治任一位点突变率[54.5%(54/99)](χ ²=95.483,P<0.05);初治患者同时耐利福平和异烟肼的耐药率(简称“耐两药率”)[2.9%(9/312)]明显低于复治耐两药率[33.3%(33/99)](χ ²=75.944,P<0.05)。利福平和异烟肼任一位点突变率为20.2%(83/411),其中利福平耐药基因ropB的突变率为5.4%(22/411),20例为单一位点突变,2例为双位点突变,以531(C→T)突变频率最高[40.9%(9/22)];异烟肼耐药基因katG和inhA的突变率为4.6%(19/411),其中13例为katG基因突变,6例为inhA基因突变,以315(G→C)突变频率最高[63.2%(12/19)];耐两药率为10.2%(42/411),其中40例为双位点突变,1例为三位点突变,1例是四位点突变,以531(C→T)315(G→C)突变频率最高[47.6%(20/42)]。结论 龙岩市肺结核耐药患者以耐两药为主,复治患者耐两药的情况较初治患者严重,防治形势严峻。     

结核分枝杆菌临床分离株耐药性与耐药基因突变关系的研究

目的分析临床分离结核分枝杆菌耐药性与耐药基因突变的关系。方法用绝对浓度法和基因测序技术检测临床分离的22株结核分枝杆菌对异烟肼(INH)、利福平(RFP)、链霉素(SM)、卡那霉素(KM)、氧氟沙星(OFLX)、对氨基水杨酸(PAS)耐药性和相应耐药基因kat G、rpo B、rpsl、rrs、gyr A、thy A的突变。结果 22株结核分枝杆菌中耐药菌株占45.5%。INH耐药率为18.2%,RFP耐药率为27.3%,SM耐药率9.1%,KM耐药率为18.2%,OFLX的耐药率为13.6%,PAS耐药率为4.5%。敏感菌株中均未检测到耐药基因突变。耐药菌株中除2株外均检测到相应耐药基因突变。结论结核分枝杆菌对抗结核药物耐药性与相关耐药基因突变密切相关。     

结核分枝杆菌对异烟肼和利福平的耐药水平与其耐药基因突变的相关性研究

目的 探讨Mtb耐药相关基因katG、inhA、oxyR-ahpC突变与对INH的耐药水平及rpoB突变与对RFP的耐药水平的关系。 方法 采用微孔板Alamar blue显色法分别检测59株耐INH的Mtb临床分离株对INH和30株耐RFP菌株对RFP的最低抑菌浓度（the minimum inhibitory concentration,MIC）,同时用直接测序法检测对INH耐药菌株katG、inhA、oxyR-ahpC和对RFP耐药菌株rpoB的突变情况。 结果 INH MIC为0.2500~1.0000 μg/ml（低水平耐药菌株）和 MIC≥2.0000 μg/ml（高水平耐药菌株）的INH耐药株,inhA启动子突变率前者高于后者［53.8% (7/13),4.3% (2/46)］,katG 315突变率前者低于后者［15.4% (2/13),76.1% (35/46)］,χ²值分别为15.57和13.48,P值均为0.000。RFP MIC为0.5000～16.0000 μg/ml和MIC≥32 0000 μg/ml的RFP耐药株,rpoB 531和526位总突变率前者低于后者［62.5% (5/8),95.5%(21/22)］,P_确切概率＝0.048。 结论 INH耐药菌株inhA启动子突变与INH低水平耐药有关,katG 315突变与INH高水平耐药有关;rpoB 531和526位突变与RFP高水平耐药有关。     

Molecular characterization of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Kazakhstan

Kazakhstan is one of the 14 countries with a high rate of morbidity due to multidrug-resistant tuberculosis (MDR TB) in WHO European region. The aim of our study was to characterize mutations associated with drug resistance to rifampicin and isoniazid in Mycobacterium tuberculosis isolates from Kazakhstan. M. tuberculosis strains were isolated from TB patients in different regions of Kazakhstan. A drug susceptibility test was performed on Lowenstein-Jensen medium using the absolute concentration method. Sequencing analysis was performed of the rpoB rifampicin resistance-determining region and the katG gene, the oxyR-ahpC intergenic region, and the inhA promoter region in 259 MDR M. tuberculosis isolates, in 51 isoniazid-resistant isolates, and in 13 rifampicin-resistant isolates. The mutational analysis revealed that the most frequent mutations associated with rifampicin and isoniazid resistance in M. tuberculosis are the substitutions at codons 531 (82.7%) and 315 (98.4%) in the rpoB and katG genes, respectively. In addition, we have found mutations with lower frequency at codon 526 (8.4%), 533 (1.5%), and 516 (1.1%) in the rpoB gene. In 6.2% of the isolates, no mutations were found in the rpoB gene. The findings of this study provide useful data for a better understanding of the mutation spectrum of isoniazid and rifampicin resistance among strains isolated from patients in Kazakhstan. Our results are also useful for the development of diagnostic tests of MDR M. tuberculosis.     

Molecular analysis of Mycobacterium tuberculosis causing multidrug-resistant tuberculosis meningitis.

SETTING: Tertiary referral hospitals in southern Vietnam. OBJECTIVE: Molecular characterisation of multidrug-resistant (MDR) tuberculous meningitis (TBM). DESIGN: Mycobacterium tuberculosis isolates from the cerebrospinal fluid (CSF) of 198 Vietnamese adults were compared with 237 isolates from patients with pulmonary tuberculosis (PTB) matched for age, sex and residential district. Isolates resistant to isoniazid or rifampicin (RMP) were sequenced in the rpoB and katG genes, inhA promoter and oxyR-ahpC intergenic regions. RESULTS: While drug resistance rates were lower in the CSF (2.5% MDR) than pulmonary isolates (5.9% MDR), the difference was not significant. The most commonly mutated codons were 531, 526 and 516 in rpoB and 315 in katG. Four novel triple mutants in rpoB were identified. CONCLUSION: RMP resistance is a good surrogate marker for MDR-TBM in this setting. However, probes directed against these three codons would have a maximum sensitivity of only 65%. A rapid phenotypic detection test may be more applicable for the diagnosis of MDR-TBM.     

Anti-drug pattern of drug-resistant Mycobacterium tuberculosis and analysis of mutation in drug-target genes

The antimycobacterial susceptibility test was performed and minimal inhibitory concentration (MIC) to drugs was determined in 98 strains of Mycobacteium tuberculosis (MTB) isolated in Tokyo from 2000 to 2003, to find which were resistant to any of the four main anti-MTB drugs, isoniazid (INH), rifampicin (RFP), streptomycin (SM), and ethambutol (EMB). 27strains of them were resistant only to SM, and 16 strains were resistant only to INH. 51 strains of them were resistant to not only INH but also other drugs. 38 strains were resistant to both INH and RFP. 19 strains were resistant to all four drugs, including 7 strains resistant to new quinolon anti-biotics also. Nucleotide or amino-acid mutations in drug resistant MTB genome were determined by DNA sequencing method. Mutation of codon 516, 526, or 531 of rpoB gene was detected in 98% of MTBs resistant to RFP. Deletion or insertion of katG gene or nucleotide mutation at regulatory region of ahpC gene was detected in MTBs highly resistant to INH. Amino acid mutation of katG gene, especially at codon 315, was detected in MTBs resistant to INH intermediate. Nucleotide mutations at regulatory region of inhA gene were detected in MTBs resistant to INH at low level. Amino acid mutation at codon 43 or 88 of rpsL gene was detected in MTBs highly resistant to SM, and nucleotide mutation at 512, 513, or 516 of rrs gene was detected in MTBs resistant to SM at low level. Amino acid mutation at codon 306 of embB gene was detected in 87% of MTBs resistant to EMB.     

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。     

Rifampicin and isoniazid resistance mutations in Mycobacterium tuberculosis strains isolated from patients in Kazakhstan.

OBJECTIVE: To analyse possible associations of specific mutations conferring rifampicin (RMP) and isoniazid (INH) resistance with Beijing and non Beijing genotype strains of Mycobacterium tuberculosis from Kazakhstan. METHOD: Genotypic analysis of 92 multidrug-resistant (MDR), 50 INH but not RMP-resistant (INHr/RMPs) and 10 fully susceptible strains of M. tuberculosis from Kazakhstan was performed. In the MDR group, 59 strains (64.1%), and within the INHr/RMPs group, 32 strains (64.0%) were classified as Beijing genotype. RESULTS: Analysis of the rpoB gene of the MDR strains revealed 10 different mutations in five codons, with rpoB codons 531 (65.2%), 526 (23.9%) and 516 (7.6%) most frequently affected. A significantly higher proportion of the rpoB S531L mutation was found among Beijing genotype strains compared with non Beijing strains (71.2% vs. 46.2%, P = 0.027). All 92 MDR isolates (100%), irrespective of their genotype, carried a mutation in codon 315 of the katG gene (S315T). However, in the INHr/RMPS control group, the S315T mutation was significantly more prevalent in the Beijing than in the non Beijing group (96.9% vs. 71.4%, P = 0.012). CONCLUSION: The high similarity of mutations supports the assumption that transmission of resistant strains is a major reason for the emergence of drug resistance in this region.     

四川地区结核分枝杆菌katG、inhA和rpoB基因突变位点耐药水平及突变频率特征分析

目的 分析基因芯片法(芯片法)检测结核分枝杆菌(Mtb)katG、inhA和rpoB基因突变位点耐药水平、突变频率特征,为临床治疗提供参考依据.方法 选取成都市公共卫生临床医疗中心2020年1月—2021年1月收治的四川地区结核病患者118例Mtb分离株,芯片法通过katG、inhA和rpoB基因突变位点检测异烟肼(H...     

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.     

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。