耐多药结核分枝杆菌耐药相关基因突变特征分析

目的了解中国耐多药结核分枝杆菌耐药相关基因的分子特征。方法对138株耐多药结核分枝杆菌和45株敏感菌的耐药相关基因inhA、katG和oxyR-ahpC间隔区(异烟肼)、rpob(利福平)、gyrA(氧氟沙星)和rrs(卡那霉素)进行序列测定,分析其基因突变特点。结果 138株耐多药结核分枝杆菌中,14.4%的菌株inhA基因发生突变,72.5%菌株的katG基因发生突变,15.9%菌株的oxyR-ahpC基因发生突变,同时考虑这3种基因,异烟肼耐药相关基因突变检出率可达90.6%;94.2%菌株的rpoB基因发生突变,74.5%菌株的gyrA基因发生突变,61.1%菌株的rrs基因发生突变,主要的突变位点为katG315(66.7%),inhA-15(9.4%),oxyR-ahpC-10(5.1%),rpoB516(13.8%),526(26.1%)和531(49.3%),gyrA90(21.6%)和94(51%),rrs1401(61.1%)。结论我国耐多药结核菌异烟肼、利福平、氧氟沙星和卡那霉素耐药相关基因最常见突变为katG315、inhA-15,rpoB531、526和516,gyrA94和90,rrs1401。     

艾滋病合并结核病患者结核分枝杆菌耐药基因突变特征分析

目的了解云南省艾滋病合并结核病患者中结核分枝杆菌(MTB)耐利福平RNA聚合酶β亚单位编码基因(rpoB)和耐异烟肼过氧化氢酶-过氧化物酶编码基因(katG)、烯酰基还原酶编码基因(inhA)突变特点。方法采用基因芯片技术,对该省208例艾滋病合并结核病患者标本进行MTB利福平耐药相关基因rpoB和异烟肼耐药相关基因katG、inhA分析。结果 208例标本中共34例(16.3%)耐药,其中单耐利福平6例(2.9%),单耐异烟肼10例(4.8%),二者同时耐药18例(8.7%)。24例利福平耐药MTB的rpoB基因突变位点主要为531(TCG→TTG),占66.7%(16/24);28例异烟肼耐药MTB的基因突变位点主要为katG基因315(AGC→ACC),占78.6%(22/28)。结论云南省艾滋病合并结核病患者中MTB利福平和异烟肼耐药基因突变具有多态性,其中耐利福平rpoB基因的主要突变位点为531(TCG→TTG),耐异烟肼katG、inhA基因的主要突变位点为katG基因315(AGC→ACC)。     

吉林省耐多药结核分枝杆菌rpoB及katG基因突变特征分析

目的分析吉林省耐多药结核分枝杆菌临床分离株rpoB、katG基因突变特点,为建立本省快速耐多药检测方法提供参考。方法对吉林省耐多药结核分枝杆菌菌株扩增rpoB、katG基因测序后比对分析。结果耐多药结核分枝杆菌rpoB、katG基因的突变率分别为83.53%和70.6%,突变类型包括点突变、联合突变和缺失。在rpoB基因中,82.30%突变发生在RRDR区域,最常见突变位点为rpoB 531、rpoB 526、rpoB 516。KatG基因突变率为70.6%,最常见突变位点为katG315,占所有突变的68.55%。结论吉林省耐多药结核分枝杆菌最常见突变位点为rpoB531、rpoB 526、rpoB 516和katG 315。     

基因芯片法用于检测长沙地区结核分枝杆菌耐药性研究

目的了解长沙市中心医院2013年1月1日至2014年9月30日临床分离的1 031株结核分枝杆菌对利福平和异烟肼的耐药性。方法将结核分枝杆菌培养阳性,并经鉴定为结核分枝杆菌的1 031株菌株,采用绝对浓度法进行常规药敏检测,同时应用基因芯片技术检测利福平和异烟肼耐药相关的rpoB、katG和inhA基因上的位点突变,并对2种结果进行比较。结果基因芯片法检测利福平耐药性中,其敏感株896株,耐药株135株;检测异烟肼耐药性为敏感株901株,耐药株130株。与绝对浓度法比较,基因芯片检测利福平耐药结果与其一致的菌株共1 011株(包含敏感株894株,耐药株117株),符合率为98.00%;基因芯片检测异烟肼耐药结果与其一致的菌株共1 005株(包含敏感株890株,耐药株115株),符合率为97.48%。与利福平耐药相关的rpoB基因的突变位点最常见的为531TCG→TTG突变,占耐药株的51.11%;其次为526CAC→TAC突变耐药株的10.37%;526CAC→GAC共11株菌株突变,占耐药菌株的8.15%。异烟肼耐药主要是由katG315AGC→ACC位突变引起,占耐药株的83.85%,inhA-15C→T突变仅占12.30%。结论基因芯片法与绝对浓度法结果高度一致,可成为筛查利福平和异烟肼耐药性的快速有效方法,长沙地区结核分枝杆菌利福平和异烟肼耐药以rpoB531,526及katG315突变位点为主。     

结核分枝杆菌对利福平和异烟肼耐药基因突变快速检测方法的建立

目的建立结核分枝杆菌对利福平和异烟肼耐药基因突变的快速检测方法。方法根据结核分枝杆菌标准株H37Rv序列，自行设计覆盖rpoB、katG、inhA基因突变区的系列寡核苷酸探针，并检测临床样品中结核分枝杆菌的基因突变情况，以此来判断耐药结果。结果在56个利福平耐药菌株中，有50个菌株都在rpoB基因上榆出有突变，利福平耐药突变检出率为89．3％（50／56）；有30个利福平敏感菌株rpoB基因上都未检出突变。有58个异烟肼培养的耐药菌株中有47个在katG或inhA基因上检出有突变，异烟肼耐药突变检出率为81．0％（47／58）；有30个异烟肼敏感菌株katG或inhA基因上未检出突变。结论用膜芯片检测结核分枝杆菌对利福平和异烟肼的耐药性，具有较高的特异性和敏感性，可用于临床结核分枝杆菌耐药性的检测。     

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.     

RIFO杂交和限制性片段长度多态性检测耐利福平和异烟肼的结核分枝杆菌

目的评价利福平寡核苷酸探针杂交技术（RIFO杂交）和PCR-限制性片段长度多态性（PCR-RFLP）在结核分枝杆菌（MTB）耐利福平（RIF）和异烟肼（INH）快速检测中的应用价值。方法选取121株北京地区MTB菌株，分别采用RIFO杂交技术和PCR—RFLP检测RIF耐药相关基因rpoB核心区和INH耐药相关基因katG315位点突变，并对所有菌株的rpoB基因核心区进行测序验证。结果RIFO杂交检测发现，91，5％（65／71）的RIF耐药株和92．9％（52／56）的耐多药菌株（至少对RIF和INH耐药）存在rpoB基因核心区突变，而RIF敏感株中未发现突变；RIFO杂交与测序结果完全一致，测序结果中有突变的位点在RIFO杂交中均有相应的野生型杂交信号缺失；PCR-RFLP结果显示，INH耐药株中katG315突变率为60.6％（40／66）。结论rpoB基因核心区可作为RIF耐药检测的分子标志及耐多药的筛选指标；RIFO杂交技术是检测MTB耐RIF的快速、准确的实验方法，具有推广及潜在的临床应用价值；PCR—RFLP可检测出大部分INH耐药株，可作为临床INH耐药性检测的辅助手段。     

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。     

尘肺并发结核患者耐多药结核分枝杆菌L型异烟肼耐药基因katG序列突变特点分析

目的探讨淮南矿区尘肺并发结核患者感染耐多药结核分枝杆菌(multiple drugs resistant Mycobacterium tuberculosis,MDR-MTB)L型异烟肼耐药基因katG突变特点。方法收集114株MTB菌L型临床分离株,用药敏试验鉴定MDR-MTB菌L型;抽提MDR-MTB L型和H37Rv标准菌株DNA,PCR法扩增katG基因,并对katG基因的突变集中区域进行测序分析。结果从114株临床分离株中检出MDR-MTB菌L型31株(27.2%),其katG基因突变率为61.3%(19/31);主要集中在315位点碱基置换突变,以Ser315Thr为主(AGC→ACC)(48.4%,15/31),其次是315位点Ser315Asn(AGC→AAC)突变(9.7%,3/31)以及431位点Ala431Val(GCG→GTG)突变(3.2%,1/31),未发现多位点联合突变,12株(38.7%,12/31)未发现katG基因突变;随机检测的10株异烟肼敏感株未见katG基因单链构象异常。结论淮南矿区尘肺并发结核患者感染的MDR-MTB菌L型异烟肼耐药情况较为严重,高度保守的katG基因突变是导致异烟肼耐药的分子基础,其突变位点呈多样性。     

四川德阳地区结核分枝杆菌耐药基因突变位点的分布特点及基因型耐药的分析

目的探讨四川德阳地区结核分枝杆菌耐药基因突变位点的分布特点及基因型耐药的分析。方法收集在2010年2月-2013年3月检出的257例肺结核病患者结核分枝杆菌DNA阳性的痰液标本,采用聚合酶链式反应（PCR）和反向点杂交（RDB）相结合的基因芯片技术对结核分枝杆菌耐药基因突变位点进行检测和分析。结果 257例肺结核患者中检测出49例存在结核分枝杆菌耐药基因位点突变,其中发生katG 315、rpsL43、embB 306和rpoB 531丝氨酸（S531L）位点突变的分别为30例（11.67%）、18例（7.00%）、11例（4.28%）和10例（3.89%）。234例初治肺结核患者对异烟肼、利福平、乙胺丁醇及链霉素的基因型耐药率分别为9.83%、4.27%、3.42%和5.13%,耐多药率为2.99%;23例复治肺结核患者对上述药物的基因型耐药率分别为52.17%、26.09%、13.04%和43.48%,耐多药率为13.04%。初治患者的基因型耐药率及耐多药率均明显低于复治患者。结论四川德阳地区结核分枝杆菌常见耐药基因突变位点分别为katG 315、rpsL 43、embB 306和rpoB 531丝氨酸（S531L）突变位点;肺结核初治患者的基因型耐药率及耐多药率均明显低于复治患者;该地区肺结核的耐多药率相对较低。     

Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent low-density DNA microarrays

BACKGROUND: A new, fast 'low cost and density' DNA microarray (LCD array), designed for the detection of mutations that confer isoniazid or rifampicin resistance in Mycobacterium tuberculosis isolates, has been developed and was evaluated using 46 resistant clinical isolates from Barcelona. METHODS: LCD chips are pre-structured polymer supports using a non-fluorescent detection principle based on the precipitation of a clearly visible dark substrate. One LCD chip consists of eight identical microarrays, designed to detect mutations within the 90 bp rpoB region, codon 315 in the katG gene and the mabA-inhA regulatory region. A total of 22 strains with a katG 315 mutation, 19 strains with alterations in the mabA-inhA regulatory region and 16 strains with mutations in the rpoB region, characterized previously, were studied. RESULTS: The identification of S315T and S315N mutations using the LCD was 100% concordant with the sequencing data. A strain with the S315R mutation, which is not tiled on the LCD array, was detected by the absence of hybridization using the wild-type probe. Of 19 strains with low-level isoniazid resistance related to the mabA-inhA regulatory region, 18 were identified correctly. The detection of mutations in the rpoB region was 93.8% concordant with the sequencing data. One mabA-inhA and rpoB mutated strain showed a cross-hybridization. CONCLUSIONS: The LCD array protocol takes 45 min (15 min 'hands-on' time) after prior PCR amplification. Only minimal laboratory equipment is required. LCD arrays provide a rapid and economical method to characterize mutations in codon 315 of the katG gene, in the mabA-inhA regulatory region and in the rpoB gene.     

浙江省丽水市结核分枝杆菌临床分离株耐药性及基因突变情况

目的 调查浙江省丽水市结核病定点医院患者结核分枝杆菌（MTB）临床分离株耐药特征及耐药基因突变情况，为该地区耐药结核病防控提供依据。方法 采用荧光PCR熔解曲线法对培养自丽水市结核病定点医院结核病患者MTB临床分离株进行抗结核药物耐药性及耐药基因突变位点检测及分析。结果 242株MTB总耐药突变占16.53%(40/242)，耐多药率为4.96%(12/242)，耐药顺位分别为异烟肼>链霉素>利福平>氟喹诺酮类>乙胺丁醇。链霉素耐药株基因突变位点主要发生在rpsL43位密码子，占比为65.00%(13/20)；异烟肼耐药基因突变位点主要发生在katG315密码子，占比为76.19%(14/21)；利福平耐药基因突变位点主要发生在rpoB基因529-533位密码子，占比为61.11%(11/18)；乙胺丁醇耐药基因突变位点主要发生在embB306位密码子，占77.78%(7/9)。结论 丽水市结核患者总体耐药突变低于全国耐药水平，但耐药模式和耐药基因突变情况仍较为复杂，需进一步加强耐药结核病监测及防控工作。     

Molecular characteristics of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates from the Russian Federation

OBJECTIVES: Three Mycobacterium tuberculosis genetic loci--rpoB and katG genes and the fabG1(mabA)-inhA operon promoter region--were studied to reveal the mutations associated with rifampicin and isoniazid resistance. METHODS: Four hundred and twelve isolates of M. tuberculosis from different regions of the Russian Federation were collected during 1997-2005. A matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based minisequencing method was used for the detection of mutations. RESULTS: Thirteen different variants of single mutations in codons 533, 531, 526, 516, 513 and 511 of the rifampicin resistance-determining region of the rpoB gene as well as the TTG insertion in the 514a position were found among the rifampicin-resistant isolates. Single nucleotide substitutions in codons 531, 526 and 516 (64.8%, 10.3% and 7.7%, respectively) were the most prevalent mutations. Codon 526 was shown to be the most variable of all. No mutations were detected in rpoB genes for 29 (10.7%) of the rifampicin-resistant isolates. 76.9% of the isoniazid-resistant isolates carried single mutations in codon 315 of the katG gene. For another 12.9% of them, double mutations in the katG gene and the fabG1(mabA)-inhA promoter region were revealed. No mutations were detected in 8.2% of the isoniazid-resistant isolates. CONCLUSIONS: Molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. The new MALDI-TOF MS-based method may be used for rapid and accurate monitoring of the spread of drug resistance.     

基因芯片技术在结核分支杆菌耐药突变基因检测中的应用

目的通过基因芯片检测系统，快速检测临床样品中结核分支杆菌耐药突变情况。方法根据结核分支杆菌标准株H37Rv序列，设计了覆盖rpoB、katG，inhA基因突变区的系列寡核苷酸探针，制作膜芯片，检测临床样品中结核分支杆菌基因突变情况，以此判断耐药结果。结果在305例临床病例中，共检出阳性病例125例，其中阳性敏感病例64例，阳性突变病例61例，阳性率为40．98％，在125例阳性样品中，共发现有8种突变类型，其中10例531L，占7．94％，19例315M，占阳性样品中总数的15．08％。结论PCR与膜芯片杂交技术可临床检测结核分支杆菌对利福平和异烟肼的耐药性，并具有快速、简便、敏感的特点。     

Molecular characterization of drug-resistant and -susceptible Mycobacterium tuberculosis isolated from patients with tuberculosis in Korea

We investigated the causal relationship between genotype and phenotype of drug-resistant Mycobacterium tuberculosis isolates obtained from patients with pulmonary tuberculosis (TB) in Korea. Of 80 isolates tested, 17, 20, 1, and 7 isolates were mono-resistant to ethambutol (EMB), isoniazid (INH), pyrazinamide (PZA), and rifampicin (RFP), respectively, and 31 isolates (38.8%) were multidrug-resistant (MDR). Sequencing analysis showed that 78% (32/41) of RFP-resistant strains had mutations in the rifampicin resistance-determining region (RRDR) of rpoB, and the mutation at rpoB531 (59.4%) was most abundant. In 52 INH-resistant strains, mutations were found mostly at C-15T (n = 21, 40.4%) in the inhA promoter region as well as at katG315 (n = 12, 23.1%). Mutations at embB306 were mostly found in 26.7% (12/45) of EMB-resistant isolates. New mutations found here in MDR isolates include rpoB523 (Gly523Glu) and embB319 (Tyr319Ser). Consequently, mutations in the rpoB531, C-15T in the inhA promoter region, embB306, and katG315 would be a useful marker for rapid detection of MDR M. tuberculosis isolates in Korea.     

Prevalence of and molecular basis for tuberculosis drug resistance in the Republic of Georgia: validation of a QIAplex system for detection of drug resistance-related mutations

We developed a QIAplex system for the simultaneous detection of 24 Mycobacterium tuberculosis gene mutations responsible for resistance to isoniazid (INH), rifampin (RIF), streptomycin (STM), and ethambutol (EMB) in 196 M. tuberculosis isolates recovered in the Republic of Georgia. In comparison to phenotypic susceptibility tests, the QIAplex showed sensitivity and specificity of 85.4% and 96.1% for INH, 94.4% and 99.4% for RIF, 69.6% and 99.2% for STM, 50.0% and 98.8% for EBM, and 86.7% and 100.0% for multidrug resistance, respectively. The dominant resistance mutations revealed were a mutation in katG resulting in S315T (katG S315T), rpsL K43R, and rpoB S531L. Mutations katG S315G and S315T and rpoB S531L were detected with higher frequencies in pretreated patients than in naive patients (P < 0.05). Simultaneous detection of 24 common drug resistance-related mutations provides a molecular tool for studying and monitoring M. tuberculosis resistance mechanism and epidemiology.     

Disequilibrium in distribution of resistance mutations among Mycobacterium tuberculosis Beijing and non-Beijing strains isolated from patients in Germany

Genotypic analysis of 103 multidrug-resistant Mycobacterium tuberculosis strains isolated in Germany in 2001 revealed that mutations in codon 531 (75.7%) of the rpoB gene and codon 315 (88.4%) of the katG gene are most frequent. Beijing genotype strains (60.2% of all isolates) displayed a different distribution of resistance mutations than non-Beijing strains.     

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.