山东地区结核分枝杆菌临床菌株基因型特征研究

目的 探讨山东地区结核分枝杆菌临床分离菌株的基因型特征,评估不同基因型分布对于耐药结核菌近期传播的影响.方法 在山东地区选取13个结核病防治机构作为监测哨点收集临床分离菌株和相关信息,应用分枝杆菌散在重复单位(MIRU)技术分析结核分枝杆菌DNA多态性.结果 1年的研究期内共获得558株结核分枝杆菌,对12个MIRU位点进行检测共产生143个基因型,其中成簇基因型66个,成簇率86.2%.74.6%的菌株属于北京家族,177个(31.7%)菌株属于山东基因型.耐多药菌株的近期感染估计值明显低于敏感菌株.结论 山东地区结核分枝杆菌具有明显的基因多态性,山东基因型菌株在人群中具有较强的传播能力.     

MIRU技术在结核杆菌基因分型中的应用

目的建立结核分枝杆菌散在分布重复单位(MIRU)基因分型法,应用于突发疫情结核病原的分型检测。方法采用简单数字表法随机抽取宁波市江北区2009-2011年保存的结核菌株,对其进行MIRU分型,同时对2011年一起学校结核突发疫情的病原进行分型检测,比较成簇性,分析传播的危险性。结果 29株江北区2009-2011年菌株MIRU分型结果分为22种不同的MIRU基因型,成簇性低;而一起学校结核突发疫情6份样本MIRU基因型分型结果为同一种MIRU基因型,成簇性高,传播危险性大。12个MIRU位点的多态性分析表明各位点有较大区别(h值为0.67～0.87之间)。结论 MIRU分型方法简单快速,其数字化结果清晰可靠,是一种有效的结核分枝杆菌基因型分型方法。应用于突发疫情病原分析,能有效控制疫情蔓延。     

我国西部贵州地区结核分枝杆菌分子流行病学初步研究

目的 分析结核分枝杆菌临床分离株的基因型特征,估计结核分枝杆菌近期在人群中传播的情况。方法 在肺结核报告发病率高的4个县级结核病门诊收集临床分离结核菌株的相关信息,应用RD105缺失基因和MIRU-VNTR基因分型技术分析结核分枝杆菌DNA多态性。结果 273株结核菌株中49.1%的菌株属于北京基因型,15 MIRU-VNTR位点组合进行分析,273株结核菌株共被分为262种不同的基因型,其中独特型251株,占91.9%,其余22株（8.1%）菌株属于11个不同的基因型,菌株成簇率为8.1%。结论 15位点组合用于本次分子流行病学研究,能准确反映贵州地区结核分枝杆菌分子流行病学特征,贵州省4个县结核分枝杆菌菌株呈现较高的多态性,其中8.1%的患者是由于近期本地区传播造成。     

基于DTM-PCR、MIRU-VNTR和耐药基因测序的武汉市耐多药结核分枝杆菌的分子分型

目的描述武汉市耐多药结核(multidrug-resistant tuberculosis, MDR-TB)分枝杆菌基因型分布及成簇特征。方法采用目标缺失多重PCR(Deletion-Targeted Multiplex, DTM-PCR)、24位点MIRU-VNTR(mycobacterial interspersed repetitive unit-variable number tandem repeat typing)以及耐药基因测序方法,对武汉市149株耐多药结核分枝杆菌进行分子分型,计算Hunter-Gaston分辨指数(Hunter-Gaston discriminatory index, HGDI),成簇率以及近期感染率最小估计值。结果全部MIRU位点的累积HGDI为0.994 4。QUB11b位点的HGDI最高(0.673 8)。联合采用DTM-PCR以及MIRU-VNTR,共鉴定出111个独立基因型和11个成簇基因型(38株),成簇率为25.50%,近期感染率最小估计为18.12%。经过耐药基因测序进一步分型,各簇内共有17(44.74%)个菌株有相同的突变位点,其余21个菌株突变位点不相同。结论联合采用DTM-PCR、24位点MIRU-VNTR分型和耐药基因测序,其基因分型分辨率较高,适合于武汉地区耐多药结核分枝杆菌的基因分型。武汉市MDR-TB的发生大多由于内源性复燃引起,但仍有一定的近期传播率。     

结核分枝杆菌MVLA基因分型成簇的影响因素分析

目的探讨马鞍山地区结核病患者结核分枝杆菌基因分型成簇的影响因素。方法采用PCR及琼脂糖凝胶电泳技术,选取15个多位点可变数目串联重复序列分析(MVLA),对104株结核分枝杆菌临床分离株进行基因分型,同时对基因分型成簇的影响因素进行分析。结果 104株结核分枝杆菌菌株共分为38个不同的基因型,成簇基因型占83.65%(87/104),单一基因型占16.35%(17/104)。耐药菌株、不同性别和不同治疗患者在成簇和单一基因型的分布上差异无统计学意义(χ2=0.036,P=0.849),但在不同年龄之间差异有统计学意义(P0.05)。结论马鞍山地区结核分枝杆菌基因型成簇率较高,提示结核分枝杆菌近期传播的风险较高,应加强对结核病患者的管理。     

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。     

武汉市耐多药结核分枝杆菌的MIRU-VNTR分子特征

目的 描述武汉市耐多药结核分枝杆菌基因型分布及成簇特征。方法 选取15个结核分枝杆菌重复序列（mycobacterial interspersed repetitive unit,MIRU）,对98株耐多药结核分枝杆菌进行多位点数目可变串联重复序列分析（mycobacterial interspersed repetitive unit-variable number tandem repeat typing,MIRU-VNTR）分型,计算差异遗传值、Hunter-Gaston指数（Hunter-Gaston discriminatory index,HGDI）、成簇率以及近期感染率最小估计值。结果 MIRU位点组合的多态性差异遗传值为0.960 4、HGDI为0.970 8,多态性和分辨指数最高的位点为Mtub 21及MIRU 26。共产生7个基因簇和65个独立基因型,成簇率为33.67%,近期感染率最小估计为26.53%。结论 15位点MIRU-VNTR分型法分辨指数较高,适合于武汉地区耐多药结核分枝杆菌的基因分型。尽管武汉市耐多药肺结核病的流行多半归因于内源性复燃,但仍有较高比例的近期传播。     

结核分枝杆菌散在重复单元分型技术在监狱结核病暴发中的应用

目的 探讨结核分枝杆菌散在重复单元在监狱结核病暴发中的应用价值.方法 应用结核分枝杆菌散在重复单位(MIRU)基因分型技术,对监狱结核病患者结核分枝杆菌分离株DNA进行基因型检测分析.结果 7例监狱结核病患者结核分枝杆菌分离株中,6例结核分枝杆菌分离株MIRU基因型为233325153533,另一分离株MIRU基因型为242325152322;6例监狱结核病患者为同一传染源直接传播的结果,而另1例患者和其他6例患者非同一传染源传播.结论 MIRU基因分型技术中结核病暴发时追踪传染源是可行的.     

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.     

宜昌地区结核分枝杆菌北京基因型菌株的鉴定及VNTR分型

 目的 研究宜昌地区结核分枝杆菌北京基因型菌株的多态性和流行特征,揭示该地区结核病的分子流行病学特征,为结核病防治提供科学依据。方法 选择2018年1月-2019年12月具有完整病例信息的结核分枝杆菌298株,进行人型结核分枝杆菌的鉴定,用扩增RD105缺失片段的多重PCR (DTM-PCR)鉴定北京基因型菌株;应用优化的9位点数目可变串联重复序列技术(VNTR)分析北京基因型菌株的多态性。结果 298株结核分枝杆菌中,260株为人型结核分枝杆菌,DTM-PCR鉴定发现北京基因型菌株140株(53.85%),非北京基因型菌株120株(46.15%)。北京基因型菌株耐药率(32.14%)高于非北京基因型菌株耐药率(10.83%),差异有统计学意义(P<0.05)。VNTR-9位点对北京基因型菌株的分辨率为0.998 5,成簇率为12.15%。结论 北京基因型菌株在宜昌地区呈较高流行趋势,且北京基因型菌株耐药率更高。VNTR-9位点基因分型方法用于该地区结核分枝杆菌北京基因型菌株的鉴别,能准确反映宜昌地区结核分枝杆菌的分子流行病学特征。     

杭州地区结核分枝杆菌氧氟沙星耐药株的MIRU-VNTR基因分型研究

目的：探讨MIRU-VNTR技术在杭州地区结核分枝杆菌氧氟沙星耐药株基因分型中的应用。方法收集杭州市2010年4月-2012年6月各结核病定点医院分离培养的临床菌株,进行药物敏感性检测。分别采用RD105缺失基因检测法和MIRU-VNTR技术对氧氟沙星耐药株进行菌株鉴定和基因分型。结果筛选出的52株氧氟沙星耐药株中,43株（82.69%）为北京家族菌株。经12个MIRU-VNTR位点组合分析,52株氧氟沙星耐药株呈52种MIRU-VNTR基因型,总Hunter-Gaston指数（ HGI）为0.999。除MIRU40和ETR-F外,其他10个MIRU-VNTR位点对北京家族菌株和非北京家族菌株均显示较高或中等程度的分辨率。结论筛选到的10个MIRU-VNTR位点具有较高分辨率,适用于杭州地区结核分枝杆菌氧氟沙星耐药株分型。     

Molecular strain typing contribution to epidemiology of tuberculosis in Limousin (1998 to 2006)

OBJECTIVES: We conducted a molecular epidemiology of Mycobacterium tuberculosis in Limousin, a French area with a low incidence of tuberculosis (4.8/100,000 inhabitants in 2005) to define the molecular diversity and the pattern of transmission. DESIGN: Two hundred and fifty-nine strains were isolated (each strain corresponds to one patient) from 1998 to 2006. Both spoligotyping and MIRU15 were chosen for our study because of their discriminatory power. RESULTS: Only 165 medical records were available: 99M/66F, mean age 56.4 years (14-94), 32.7% foreign-born patients, 16.9% homeless or living in shelters, 21.8% of immunocompromised patients (three HIV positive), 14.5% of alcohol addicts. Pulmonary manifestations were predominant (81.8%) with 45.1% of positive smears. Two strains among the 259 presented a multidrug resistance. Spoligotyping identified 136/259 spoligotypes (110 unique, 26 clusters composed of two to 36 isolates); within these 26 clusters, ST53 (n=36) and ST50 (n=19) were the most frequent. Three major families were observed as follow: T1 (30%), Haarlem (30%) and LAM (20%). MIRU15 identified 28/36 isolates in the ST53 group and 14/19 in the ST50 group. Eleven clusters (32 strains) with identical ST-MIRU15 were obtained with a proved case of recent transmission. Alcohol dependence, immunosuppression and pulmonary infections seem to be involved in transmission factors. CONCLUSION: M. tuberculosis strains isolated in Limousin are characterized by their high genetic diversity. The rate of recent transmission (8.1%) is low and therefore a reactivation process is predominant in this area.     

24-locus MIRU-VNTR and Spoligotyping analysis of drug-resistant Mycobacterium tuberculosis strains isolated from Northeastern Thailand

Tuberculosis, caused by Mycobacterium tuberculosis (MTB) infection, remains a global health problem with increased concerns due to drug-resistant tuberculosis. However, molecular genotyping profiles may give insight of the transmission of TB in a particular region. The present study aimed to characterize the genetic diversity of drug-resistant MTB and evaluate primer sets applied for the epidemiological study of circulating MTB in Northeastern Thailand. A total of 92 MTB isolates, resistant to rifampicin and/or isoniazid, were collected from the Office of Disease Prevention and Control between 2013 and 2016. All isolates were genotyped by 24-locus MIRU-VNTR typing combined with spoligotyping. We also analyzed the distributions of drug susceptibility pattern and demographic data among different genotypes. In comparison with different loci sets, discriminatory power based on 12, 15, 24 standard primers were investigated. Eighty-six particular profiles were found; among the patterns, two clusters were produced in 8 strains. East African Indians (EAI) were the most prevalent strains (33 isolates, 35.87%) followed by Beijing (30 isolates, 32.61%), with 23 unknown isolates strains also found. The HGDI based on combination of 24 loci analysis and spoligotyping was 0.9962. The number of tandem repeat generated was highly discriminant (HGDI>0.6) at locus 580 (0.66), 960 (0.67), 2163b (0.73), 2165 (0.62), 2461 (0.68) 3690 (0.73) and 4052 (0.79), respectively. In contrast, the diversity at locus 154 and 2059 was not revealed. The results emphasized that 24-locus MIRU-VNTR and spoligotyping could be useful for epidemiological surveillance of drug-resistant MTB in this region. At a given allelic diversity, 7 primer sets containing MIRU04, MIRU10, QUB2163b, ETRA, ETRB, Mtub39 and QUB26 may be considered for screening the VNTR patterns. In addition, this study gathered both demographics and genotypic data within the same investigation for further tuberculosis prevention and control.     

13个VNTR位点用于113株结核分支杆菌的基因分型研究

目的 应用数目可变串联重复序列（VNTR）分子分型技术，对北京地区113株肺结核临床分离菌株进行分型研究，探讨北京地区菌株DNA多态性和基因型特征。方法 采用PCR和琼脂糖凝胶电泳技术对结核分支杆菌13个VNTR位点进行检测，并应用Gel-Pro analyzer 3．1软件和BioNumerics3．0软件进行结果分析。结果 113株结核分支杆菌可分为4个基因型（分别为Ⅰ、Ⅱ、Ⅳ和Ⅴ型），其中Ⅰ型占92．0％（104/113），其他3型所占比例很小，分别为Ⅱ型占4．4％（5／113），Ⅳ和Ⅴ型均为1．8％（2／113），而标准菌株H37Rv在分型中为独立的一个基因型，即Ⅲ型。结论 北京地区的结核分支杆菌存在基因多态性，其主要流行型为Ⅰ型。     

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.     

Differentiation of tuberculosis strains in a population with mainly Beijing-family strains

A high prevalence of tuberculosis (TB) isolates that are genetically homogenous and from the Beijing family has been reported in Russia. To map TB transmission caused by these strains, new genotyping systems are needed. Mycobacterial interspersed repetitive units (MIRUs) offer the possibility of rapid PCR-based typing with comparable discrimination to IS6110 restriction fragment length polymorphism techniques. Spoligotyping and detection of IS6110 insertion in the dnaA-dnaN region were used to identify Beijing strains in 187 Mycobacterium tuberculosis isolates from Samara, Russia. The Beijing isolates were analyzed by using 12-MIRU and 3-exact tandem repeats (ETR) loci and by an expanded set of 10 additional variable number tandem repeats loci. The expanded set of 25 MIRUs provided better discrimination than the original set of 15 (Hunter-Gaston diversity index 0.870 vs. 0.625). Loci MIRU 26, 1982, and 3232 were the most polymorphic in Beijing isolates.