福建省耐多药结核分枝杆菌耐药相关基因突变特征

目的初步分析福建耐多药结核病(multi-resistant tuberculosis,MDR-TB)临床分离结核分枝杆菌利福平和异烟肼的耐药相关基因突变情况。方法自福建福州肺科医院收集耐多药结核分枝杆菌临床分离菌株,采用PCR测序技术测定利福平和异烟肼耐药相关基因,包括rpoB、katG、inhA和mabA-inhA,并与标准菌株基因序列比对,分析基因突变特点。结果共对67株MDR-TB菌株进行了分析,91.04%的菌株在rpoB81 bp RRDR区发生突变,其中,88.06%(59/67)的MDR-TB菌株在rpoB531、526和516位发生突变;79.10%的菌株在katG、inhA或mabA-inhA上发生突变,59.70%(40/67)的菌株在katG315或者mabA-inhA(-15)位发生突变,没有检测到同时在inhA结构基因和基因调节区发生突变的菌株。结论 PCR-DNA测序分析技术可以用于检测MDR-TB临床分离菌株对RFP和INH的药物敏感性。     

华东农村地区耐药结核病传播及其影响因素的分子流行病学研究

目的 对江苏和浙江省两个农业县全年登记的结核病患者中分离获得的耐药结核分枝杆菌(结核菌)进行基因分型,描述以基因型"成簇性"为定义的耐药结核病在该地近期传播及其影响因素.方法 以结核菌散在分布重复单位(MIRU)和IS6110限制性内切酶片段长度多态性(IS6110-RFLP)基因分型技术识别耐药结核菌基因型的"成簇性"和"惟一性",描述223例耐药结核病患者结核菌分离株的成簇性分布特征,识别耐药结核菌的人群间传播,分析人口学特征、社会经济水平和就医行为对耐药结核病传播的影响.结果 223株耐药结核菌株中有52株具有成簇性,形成22个簇,提示簇内患者间发生了耐药结核病的近期传播;其余171株的基因型表现为"惟一性".耐药结核菌成簇性影响因素分析显示:与18～30岁人群相比,30～60岁年龄组(30～岁/18～岁年龄组:30.9% vs.11.9%;OR=3.297,95%CI:1.169～9.297)和复治耐药结核病患者(复治/初治:32.9% vs.18.4%;OR=2.163,95%CI:1.144～4.090)所感染的结核菌更容易成簇;对异烟肼和利福平同时耐药的耐多药结核菌比仅耐一种一线抗结核药物的单耐药菌株更多地表现为成簇(47.2%vs.15.5%;OR=4.773,95%CI:2.316～9.837),耐药结核菌株的近期传播呈现以村落为单位的小范围、散发特点.结论 华东农村地区人群中存在耐药结核病的近期传播,其主要传播方式可能为村落内偶然接触;耐药结核病的控制需要重点关注中年人群和复治结核病患者.     

基于全基因测序的宝安区耐药结核菌株基因突变及其特征分析

目的 分析宝安区耐药结核菌株基因突变及其特征,为耐药结核病防控提供支撑。方法 以全基因测序结果为基础,采用[AKx-]±s或频数和构成比等描述性统计分析方法,回顾性分析2015—2019年宝安区耐药菌株基因突变情况。结果 2015—2019年,宝安区结核病总体耐药率为18.45%（388/2 103）,单耐药菌株占比接近60%（225/388）,其中单耐异烟肼和利福平菌株的比例超过50%（118/225）。耐药菌株中,对链霉素产生的耐药性突变菌株占比为44.59%（173/388）,主要突变位点发生在rpsL基因的43位（59.54%,103/173）;对异烟肼产生的耐药性基因突变位点发生在katG基因的315位（68.21%,103/151）,且发现9例菌株发生联合突变,1例菌株发生移码突变;对利福平产生耐药性基因突变主要发生在rpoB基因的450位（38.60%,44/114）,13例菌株发生了联合突变（11.40%,13/114）。结论 宝安区耐药结核分枝杆菌以L2型、L4型为主,对链霉素、异烟肼和利福平产生耐药性基因突变位点主要发生在rpsL基因的43位、katG基因的315位和rpoB基因的450位,且对异烟肼、利福平发生了联合性耐药基因突变。     

PCR-SSCP检测结核分枝杆菌利福平耐药性

目的:建立PCR-SSCP快速检测结核分枝杆菌利福平耐药相关基因rpoB突变。方法:用比例法筛选出70株耐利福平结核分枝杆菌临床分离株,用PCR-SSCP方法检测菌株ropB基因突变,并与比例法检测结果进行对比。结果:70株耐利福平菌株中,单耐利福平菌株为12株,同时耐福平和异烟肼菌株为58株。扩增菌株rpoB基因,70株结核分枝杆菌临床分离株ropB基因突变率为91.43%(64/70),其中,耐多药菌株ropB基因突变率为91.38%(53/58),单耐利福平菌株ropB基因突变率为91.67%(11/12)。结论:用PCR-SSCP技术检测结核分枝杆菌RFP耐药性所用时间大幅缩短,检测出的结果与传统方法一致率高,但结核分枝杆菌表型耐药菌株,存在rpoB基因位点无突变的现象,是否存在其他耐药位点或耐药机制,值得深入研究。     

淮南矿区煤工尘肺结核患者耐多药结核分枝杆菌耐药基因rpoB序列分析

目的 分析淮南矿区尘肺结核患者感染的耐多药结核分枝杆菌(multiple drugs resistant bacillus tuberculosis,MDR-TB)利福平耐药基因rpoB突变特点.方法 收集结核分枝杆菌临床分离株114株,采用药敏试验鉴定MDR-TB,抽提MDR-TB DNA和H37Rv标准菌株DNA,PCR法扩增rpoB基因,并应用全自动DNA测序仪对rpoB基因的突变集中区域进行测序分析.结果 从114株结核分枝杆菌临床分离株中检出MDR-TB31株(27.19％,31/114),31株MDR-TB中rpoB基因突变率为93.55％(29/31),主要集中在531(45.16％,14/31)和526位点(29.03％,9/31)碱基置换突变,其次是516(3.23％,1/31)、535位点(3.23％,1/31);1株526位和531位同时突变,1株511位和526位同时突变,2株未发现rpoB基因突变.随机检测的10株利福平敏感株未见rpoB基因单链构象异常.结论 高度保守的rpoB基因突变是导致尘肺结核患者MDR-TB利福平耐药的分子基础,其突变位点呈多样性.     

西安市结核病患者耐药情况及耐药菌株基因突变现状

目的了解西安市结核病患者耐药情况,以及对常用抗结核药耐药的结核分枝杆菌(MTB)的相关耐药基因突变特征进行分析。方法对2020年1—12月西安市结核病定点医院上报的痰涂片阳性结核病患者的痰样本进行分离培养,对鉴定为MTB阳性的菌株样本进行药敏试验,并利用全基因组测序(WGS)对耐药菌株的耐药相关基因突变特征进行分析。结果共鉴定出892例MTB阳性患者,其中初治患者758例,复治患者134例;耐药209例,总耐药率23.43%;单耐药(MR-TB)、多耐药(PDR-TB)、耐多药(MDR-TB)、广泛耐药(XDR-TB)和利福平耐药(RR-TB)率分别为10.43%、2.47%、4.60%、0.45%和6.28%。复治患者的耐药率(31.34%)明显高于初治患者(22.03%);且2组耐药患者MR-TB、PDR-TB、MDR-TB、XDR-TB和RR-TB所占比例的差异均有统计学意义(均P<0.05)。209例MTB耐药菌株对常用抗结核药品的耐药相关基因突变主要为81株katG(77.14%,异烟肼)、54株rpoB(96.43%,利福平)、49株rpsL(84.48%,链霉素)、24株embB(88.89%,乙胺丁醇)、17株pncA(89.47%,吡嗪酰胺)、10株gyrA(90.91%,氧氟沙星)、18株rss(94.74%,二线注射类抗结核药物)。其中相关耐药基因突变位点频率最高的分别为katG S315T(72株)、rpsL K43R(42株)、rss A1401G(23株)、rpoB S531L(19株)、embB M306V(13株)和rpoB H526D(11株)。结论西安市结核病患者对常用抗结核药具有较高的耐药性,耐药菌株基因突变以katG、rpoB、rpsL、embB和pncA等基因突变为主,且主要发生在katG S315T、rpsL K43R等高频突变位点,针对上述突变特征开发敏感、特异的快速分子检测方法具有理论可行性。     

158株结核分枝杆菌北京基因型的耐药特征研究

我国是结核病高耐药国家,而高耐药率给结核病患者的治疗带来很大困难,并对其流行产生严重影响.本研究同时采用IS6110-RFLP、spoligotyping对结核分枝杆菌进行基因分型,探讨不同地区不同基因型的耐药特点及北京基因型与4种药物[利福平(RFP)、异烟肼(INH)、链霉素(SM)、乙胺丁醇(EM8)]耐药的关系.     

47株结核分枝杆菌利福平耐药决定区基因突变特征的研究

目的了解吉林省结核分枝杆菌的利福平耐药决定区(RRDR)基因突变特征。方法结核分枝杆菌(MTB)分离自2008-2009年吉林省临床肺结核病人的痰液标本,培养方法为改良酸性罗氏(L-J)培养基。分离出的抗酸杆菌经对硝基苯甲酸(PNB)培养基初步鉴定后选出结核分枝杆菌复合群并应用比例法进行药敏试验(DST)。根据DST结果,选择22株利福平耐药株、22株利福平敏感株以及3株临界值标本进行聚合酶链式反应(PCR)扩增rpoB中包含RRDR在内的部分基因,阳性DNA片段通过基因测序以及Bioedit软件分析方法比对基因突变情况。结果 22株利福平耐药株中,16株同时对异烟肼耐药;22株利福平敏感株中,1株异烟肼耐药。利福平耐药菌株中,95%(21/22)的菌株在利福平耐药决定区RRDR发生了基因突变,其中第531位密码子突变频率占50%,第526位占27%,第533位占9%,第516位及513位均为4.5%;22株利福平敏感株中,RRDR区全部无基因突变发生;3株临界值标本中,1株在第531位密码子发生了基因突变,1株在533位发生基因突变,最后1株无突变。结论 RRDR区点突变的发生与利福平耐药高度一致;异烟肼耐药与利福平耐药高度相关。     

江苏省结核分枝杆菌耐药相关基因突变特征研究

目的探讨江苏省结核分枝杆菌耐药相关基因的基因型特点及其与耐药表型的关系,为开展耐药结核病快速诊断提供依据。方法通过基因测序技术,分析结核分枝杆菌利福平(rifampin,RFP)耐药相关基因rpoB和异烟肼(isoniazid,INH)耐药相关基因katG不同位点的突变频率,探讨基因型与耐药表型的关系。结果共收集新登记涂阳病人菌株283株,其中255株结核分枝杆菌成功测序,包括38株RFP耐药株,50株INH耐药株,48株链霉素耐药菌株,21株乙胺丁醇耐药株,34株耐多药菌株。28株(11.0%)在rpoB 81bp的基因核心区发生了点突变,21株出现单个位点突变外,7株出现两位点突变,突变形式主要为rpoB531 TCG→TTG单个位点突变。30株(11.8%)发生了katG基因区的突变,所有突变形式为katG315AGC→ACC。rpoB基因突变全部发生于RFP耐药菌株中,katG基因突变全部发生于INH耐药菌株中。结论检测rpoB与katG基因位点的突变对判断江苏省地区耐药结核以及耐多药结核有重要的价值,可以为开展耐药结核病早期诊断提供科学依据。     

rpoB基因突变与结核分枝杆菌利福平耐药相关性研究

目的 探讨临床患者痰标本中分离的结核分枝杆菌rpoB基因突变与利福平耐药之间的关系. 方法 对115株结核分枝杆菌应用L-J药敏培养法,检测对利福平、异烟肼、乙胺丁醇、链霉素、氧氟沙星的耐药情况;根据rpoB基因序列设计包含利福平耐药决定区（RRDR）的扩增引物,PCR法进行扩增,运用生物信息学方法对耐药决定区扩增产物序列进行比对分析,对耐药株与非耐药株基因突变率进行统计学分析. 结果 115株结核分枝杆菌经比例法药敏试验,RIF耐药株53株,占所有菌株的46.09％;53株RIF耐药株中39株rpoB基因存在不同位点的变异,突变率为73.58％,突变位点包括531、526、522、516、513、511,最主要的突变位点集中在531位,引起氨基酸呈Ser531Gln、Ser531Leu、Ser531Trp 3种形式改变;RIF敏感株62株,其中有2株检测到rpoB基因的突变,敏感株与耐药株突变率经McNemar统计检验,差异有统计学意义（P＜0.05）. 结论 岳阳地区rpoB基因变异与利福平耐药呈高度相关,rpoB基因最主要的突变位点位于531位和526位.     

Role of spoligotyping and IS6110-RFLP in assessing genetic diversity of Mycobacterium tuberculosis in India

In the present study, genetic diversity analysis of Mycobacterium tuberculosis isolated from patients attending a tertiary care hospital, North India, has been attempted. Eighty three isolates of M. tuberculosis were subjected to DNA fingerprinting using spoligotyping and IS6110-RFLP techniques. Spoligotype patterns showed that central Asian (32.5%), ill defined T (13.2%) and Beijing (10.8%) families were predominant in ongoing transmission of the bacterium. Two STs; ST26 (CAS_Delhi) and ST1 (Beijing) represented 36.1% of the total M. tuberculosis population in eastern Uttar Pradesh, North India. IS6110 RFLP analysis showed that isolates having low and zero copy number of the IS element were 15.6% and 19.2%, respectively. Out of the 47 isolates clustered by spoligotyping, 40 could be further differentiated as unique strains by IS6110-RFLP. Therefore, this study recommends that both the techniques be used simultaneously for DNA fingerprinting of M. tuberculosis in India.     

Molecular epidemiology of tuberculosis in the Czech Republic, 2004: analysis of M. tuberculosis complex isolates originating from the city of prague, south Moravia and the Moravian-Silesian region

OBJECTIVES: To compare M. tuberculosis complex genotypes from representative regions of the Czech Republic in order to estimate changes in strain prevalence and in the extent of imported drug-resistant tuberculosis. METHODS: Primary M. tuberculosis complex isolates (n=155) and follow-up isolates (n=15) from 155 patients from the first half of 2004 (98 from Prague, 37 from South Moravia and 35 from the Moravian-Silesian region) were genotyped by IS6110-RFLP, spoligotyping, and partly by VNTR-genotyping. RESULTS: Based on IS6110-RFLP, 110 of 155 (71%) primary isolates were unique. Forty-five isolates (29%) were found in 15 clusters comprising two to six patients and all but one cluster were also discriminated by MIRU-VNTR-genotyping. Four clusters comprised patients from different regions, and six were ongoing for several years. An indication of MDR-strain transmission was found in one instance. All four Beijing-type isolates with any resistance were associated with immigration from Eastern Europe. CONCLUSIONS: The molecular epidemiological data of this period-prevalence, population based study and its comparison to earlier investigations point to a low extent of clustering between M. tuberculosis complex isolates in representative regions of the Czech Republic. Few clusters extending geographically and/or over several years were identified, providing a means for an in-depth analysis of risk factors of transmission. Beijing genotype isolates were shown to increase in prevalence to reach 6.5%. Drug resistant isolates of this genotype were associated with immigration of from Eastern Europe, although direct transmission of a resistant isolate was probable only in one of eleven cases.     

Interpreting genotype cluster sizes of Mycobacterium tuberculosis isolates typed with IS6110 and spoligotyping

Molecular techniques such as IS6110-RFLP typing and spacer oligonucleotide typing (spoligotyping) have aided in understanding the transmission patterns of Mycobacterium tuberculosis. The degree of clustering of isolates on the basis of genotypes is informative of the extent of transmission in a given geographic area. We analyzed 130 published data sets of M. tuberculosis isolates, each representing a sample of bacterial isolates from a specific geographic region, typed with either or both of the IS6110-RFLP and spoligotyping methods. We explored common features and differences among these samples. Using population models, we found that the presence of large clusters (typically associated with recent transmission) as well as a large number of singletons (genotypes found exactly once in the data set) is consistent with an expanding infectious population. We also estimated the mutation rate of spoligotype patterns relative to IS6110 patterns and found the former rate to be about 10-26% of the latter. This study illustrates the utility of examining the full distribution of genotype cluster sizes from a given region, in the light of population genetic models.     

Multidrug-resistant tuberculosis in prison inmates, Azerbaijan.

In a tuberculosis (TB) program in the Central Penitentiary Hospital of Azerbaijan, we analyzed 65 isolates of Mycobacterium tuberculosis by IS6110-based restriction fragment-length polymorphism (RFLP) and spoligotyping. From 11 clusters associated with 33 patients, 31 isolates had an IS6110-based banding pattern characteristic of the Beijing genotype of M. tuberculosis. In addition, 15 M. tuberculosis isolates with similar RFLP patterns constituted a single group by spoligotyping, matching the Beijing genotype. Multidrug resistance, always involving isoniazid and rifampin, was seen in 34 (52.3%) of 65 isolates, with 28 belonging to the Beijing genotype.     

Utility of gene analysis for evaluation of the current status of tuberculosis developing long after primary infection

PURPOSE: The purpose of this study was to examine the utility of gene analysis of Mycobacterium tuberculosis as a means of determining the current status of tuberculosis developing in patients long after primary infection and identifying the source of infection. METHODS: We analyzed two outbreaks of tuberculosis involving chronic carriers in 2004 within Okayama prefecture. DNA was extracted from Mycobacterium tuberculosis stains isolated from the patients, and subjected to IS6110-RFLP and PGRS-RFLP analyses. The resulting IS6110-RFLP patterns were compared with a database (compiled since December 1999) of RFLP patterns of isolates from newly registered tuberculosis patients in the prefecture. Mutations in the rpoB gene for rifampicin resistance and the katG gene for isoniazid resistance ofMycobacterium tuberculosis were detected by real-time PCR followed by melting curve analysis. Bacterial isolates were tested for antimycobacterial susceptibility by the microdilution susceptibility method and the proportion method on Ogawa's medium. RESULTS: In outbreak 1, an epidemiological survey suggested that two individuals contracted tuberculosis from the same patient after an interval of about 20 years. They differed from the suspected patient in the drug susceptibility patterns of bacterial isolates and mutations in the drug susceptibility-related genes, but the results of IS6110-RFLP and PGRS-RFLP analyses supported a conclusion of a common source of infection. In outbreak 2, a hospital employee developed primary multidrug-resistant tuberculosis of unknown origin. Comparison between the IS6110-RFLP patterns of the employee's bacterial isolate and the IS6110-RFLP database showed identity to a bacterial isolate from a chronic carrier who had died in the same hospital 4 years and 8 months earlier. Moreover, the two isolates were identical regarding drug susceptibility patterns and mutations in drug resistance-related genes, suggesting a nosocomial infection. CONCLUSION: These results indicates that tuberculosis gene analysis provides useful information for comprehension of the current status of tuberculosis developing in patients long after primary infection. Genotype database construction may allow detection of the source of infection, which cannot be identified by contact examination alone.     

IS6110限制性片段多态性分析标准方法的建立及其在结核分枝杆菌分子分型中的应用

目的 建立IS6110限制性片段多态性分析(IS6110-RFLP)标准方法 并评价该方法 的分型能力.方法 采用核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等技术,结合Gel-Pro analyzer 3.1和BioNumeries(Version 5.0)软件,对78株结核分枝杆菌插入序列IS6110-RFLP进行分析.结果 确定标准化的IS6110-RFLP技术,包括核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等实验步骤及标化参数的相关数据分析软件的使用;采用该技术,将78株结核分枝杆菌分为75个不同的基因型,分别归属于11个基因簇,其中有52株归属于同一个基因簇,占菌株总数的66.7%(52/78).结论 建立标准化的IS6110-RFLP技术方案,该方法 具有很强的基因分型和株水平鉴定能力,可用于结核病的病原学监测.     

三种不同的DNA分型技术在158株结核杆菌研究中的应用

目的评价IS6110限制性片段长度多态性(RFLP)、间隔区寡核苷酸分型(Spoligotyping)及分枝杆菌散在重复单位(MIRU)三种分型方法在结核病流行病学研究中的应用。方法对158株结核分枝杆菌临床分离株应用IS6110RFLP、Spoligotyping及MIRU三种分型方法进行鉴定。结果应用三种分型方法产生的类型数分别为118、20和105个。IS6110RFLP的分辨率大于Spoligotyping,MIRU的分辨能力与IS6110RFLP接近。在MIRU的12个区中,重复区4、10、26、40具有较高的多态性。广东地区与其他地区成簇率和北京基因型所占比例差异有统计学意义(P<0.05),广东地区成簇率和北京基因型所占比例均显著低于其他地区。结论应用IS6110RFLP、Spoligotyping及MIRU三种分型方法进行结核病流行病学研究具有重要意义且非常有效,可以发现中国不同地区菌株的不同特点。     

Rifampin- and multidrug-resistant tuberculosis in Russian civilians and prison inmates: dominance of the beijing strain family

Consecutive patient cultures (140) of Mycobacteriium tuberculosis were collected from five Russian civilian and prison tuberculosis laboratories and analyzed for rifampin (rpoB) and isoniazid resistance (inhA, katG, ahpC); transmission of Beijing family isolates; and the importance of prison and previous therapy in drug resistance. Rifampin, isoniazid, and multidrug resistance occurred in 58.2%, 51.6%, and 44.7% of cultures, respectively; 80% of prison cultures were rifampin resistant. Spoligotyping and variable number tandem repeat (VNTR) fingerprinting divided the isolates into 43 groups. Spoligotyping demonstrated that a high proportion (68.1%) of patients were infected with Beijing family strains and that most (69.1%) were rifampin resistant; the highest proportion (81.6%) occurred in prison. One VNTR subgroup (42435) comprised 68 (72.3%) of the Beijing isolates with a small number of IS6110 types; 50 (73.5%) were rifampin resistant. Rifampin-resistant Beijing isolates are dominant within the patient population, especially among prisoners, and threaten treatment programs.     

Genetic diversity, population structure and drug resistance of Mycobacterium tuberculosis in Peru

This paper presents the first evaluation of the molecular epidemiology of Mycobacterium tuberculosis in Peru. We characterised 323 isolates using spoligotyping and mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTR) typing. We aimed to determine the levels of genetic diversity and genetic differentiation among and within Peruvian isolates and the epidemiological factors which may be driving patterns of population structure and evolution of M. tuberculosis in Peru. Our results compared to the fourth international spoligotyping database (SpolDB4) and MIRU-VNTRplus, show that the main M. tuberculosis families present are Latin American-Mediterranean, Haarlem, T, and Beijing. Bayesian clustering recovered 15 groups in the Peruvian M. tuberculosis isolates, among which two were composed mainly of orphans, implying the presence of native "Peruvian" strains not previously reported. Variable levels of association with drug resistance were observed, with Beijing genotypes not showing any association with multidrug resistance, while in other groups MIRU-VNTR loci 2, 23, 31, and 40 were found to be associated with the multidrug-resistant tuberculosis (MDR-TB) phenotype, suggesting that a linkage disequibrium between these MIRU and drug resistance loci may be present. Genetic differentiation was present among drug resistant and sensitive strains. Ethambutol appeared to be the main driver of differentiation, suggesting that strong selection pressure could have been exerted by drug treatment in Peru over recent years.