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.     

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.     

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。     

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。     

Efficient Differentiation of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Strains of the W-Beijing Family from Russia using Highly Polymorphic VNTR Loci

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen’s University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment length polymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter–Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a “gold standard” method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor profiles correlation (0.767) reflects the differences in the rate and type of evolution between genome regions targeted by IS6110-RFLP and VNTR typing. VNTR typing in proposed format is powerful tool for discrimination of M. tuberculosis strains with different level of genetic relationship.     

不同串联重复序列位点组合用于中国结核分枝杆菌基因分型的能力比较分析

目的 初步评价不同串联重复序列(VNTR)位点在中国8省市结核分枝杆菌基因分型中的应用,寻找适合中国地区结核分枝杆菌基因分型的位点组合.方法 从中国8个省(市、自治区)2800余株结核分枝杆菌临床分离菌株中以简单数字表法随机抽取140株,采用多位点数目可变串联重复序列分析方法(MLVA)对27个数目可变VNTR位点进行基因多态性检测,采用BioNumerics数据库软件进行单位点和不同位点组合的分辨率(Hunter-Gaston指数,HGI)分析,并比较分析其对140株菌的基因分型鉴定能力.同时采用间隔区寡核苷酸分型(Spoligotyping)将140株菌分为北京家族和非北京家族,评价上述不同VNTR位点组合在北京家族和非北京家族中的分型能力.结果 140株菌主要可分为2个基因群,即北京家族112株,占80%;非北京家族28株,占20%.Spoligotyping分型对140株结核分枝杆菌的HGI为0.4589.MLVA分析结果显示不同位点在不同菌株群存在明显的多态性,不同位点的HGI具有较大差异(0～0.809),对全部菌株、北京家族菌株、非北京家族菌株的HGI达到0.5以上的VNTR位点数分别为8、7和14个.27个VNTR位点进行不同的位点组合:优化筛选的8位点组合、国际推荐的12个、15个和24个位点组合.4个组合的HGl分别为0.9991、0.9882、0.9980和0.9986;在北京家族菌株中,上述组合的HGI依次为0.9987、0.9318、0.9969和0.9975;在非北京家族菌株中分别为1、0.9894、1和1.结论不同的VNTR位点和不同VNTR位点组合在中国8省市结核分枝杆菌中的HGI均存在明显差异;本研究优化的8个位点组合MLVA分型方法在中国结核分枝杆菌流行病学研究可能具有良好的应用前景.     

A first insight into the application of high discriminatory MIRU-VNTR typing using QIAxcel technology for genotyping Mycobacterium bovis isolated from the Delta area in Egypt

Mycobacterium bovis is a notorious infectious agent leading to serious economic losses for cattle farms worldwide. Analysis of the widely spreading genotypes is vital for tracing infections, understanding transmission dynamics, and controlling the cluster growth. This study aimed to evaluate the discrimination ability of 15 mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR) loci and to assess the extremely efficient loci subset for molecular epidemiological investigations of M.bovis from farms in the Delta area of Egypt. The discriminating ability of MIRU-VNTR genotyping using 15 loci {2 exact tandem repeat (ETR) loci, 6 MIRU loci, 4 Mtub loci, and 3 Queen's University of Belfast (QUB) group loci)} were evaluated on 61 M.bovis isolates from cattle (Holstein Frisian) and buffaloes. The results indicate that there are 48 genotypes: 3 unique genotypes and 45 genotypes with shared similarities. Using the MIRU-VNTRplus database, M.bovis ID 7540/01 and ID 5346/02 were the nearest lineages to both groups. Six loci (MIRU10, QUB11b, QUB26, ETRA, Mtub30, and Mtub39) were highly discriminating, seven other loci (Mtub21, MIRU26, QUB4156, MIRU04 (ETRD), MIRU16, MIRU 40, and ETRC) gave moderate discriminatory power, and the last two loci (Mtub04 and MIRU31) were poorly discriminative. MIRU-VNTR typing generally proved efficacy and high discriminatory power, with a collective allele's diversification of 0.9641. Both the six highly discriminating (DI = 0.9492) and the seven moderately discriminating loci (DI = 0.9269) evidenced to be suitable for M.bovis first-step initial genotyping from cattle herds in Egypt. MIRU-VNTR is rapid and effective in the genotyping of M.bovis from cattle and buffaloes in Egypt.     

结核分枝杆菌多位点可变数目串联重复序列分型方法标准化操作程序的建立

目的 建立结核分枝杆菌多位点可变数目串联重复序列分析(MLVA)的标准化方法,评价该方法的分型能力、应用价值.方法 选取15个位点,采用核酸提取、PCR和琼脂糖凝胶电泳等技术,结合BioNumeries(Version 5.0)软件,对中国54株结核分枝杆菌临床分离株进行分型.结果确定标准化的MLVA方法,包括细菌分离培养、核酸提取、PCR、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.VNTR位点确定为ETRA、ETRB、ETRC、ETRD、ETRE、MIRU10、MIRU16、MIRU23、MIRU26、MIRU27、MIRU39、MIRU40、Mtub21、Mtub30和Mtub39.结论建立MLVA标准化技术方案,该方法操作简便,分型鉴定能力及实验室间结果可比性强,便于网络化,有利于结核病流行中追溯传染源,分析流行趋势.     

Molecular typing of Mycobacterium tuberculosis isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing.

Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB). This study was done to characterize prevalent genotypes of M. tuberculosis on a collection of 97 isolates based on spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in rural area of Kanpur, North India. In this area different types of interventions are being undertaken and follow-up studies are progressing. Predominant spoligotypes prevalent in this region belonged to Central Asian-Delhi family (CAS1_Del) (37%), East African-Indian family (11%), T1 family (8%) and Beijing (4%) family. Highly distinct MIRU-VNTR genotypes were obtained. Significant spoligotypes such as Beijing and CAS1_Del type were further divided into subtypes with MIRU-VNTR. This preliminary study reveals that CAS is the most predominant family in this rural area of Kanpur. If confirmed in other areas, this combined approach of molecular typing can be preferably be used as first line tool for studying linkage and transmission dynamics of TB in India.     

Genetic diversity of multidrug-resistant Mycobacterium tuberculosis isolates in Punjab,Pakistan

Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries and 6th among the countries with the highest prevalence of drug-resistant TB. However, insufficient data are available on the genetic structure of M. tuberculosis strains circulating in this country. The objective of this study was to explore the genetic diversity of multidrug-resistant M. tuberculosis isolates from Punjab, Pakistan with a combination of spoligotyping and 24-loci MIRU-VNTR typing.Among a total of 127 MDR isolates studies, 53 spoligotypes were obtained, split into 14 clusters (n = 88, 69.3%, 2–29 isolates per cluster) and 39 (30.7%) unique patterns. At the phylogenetic level, the most prevalent sublineage was CAS1_DELHI (n = 53, 41.7%), mostly represented by ST 1942 (n = 29, 22.8%), followed by T1 (n = 14, 11%) and Beijing (n = 10, 7.8%). The remaining nine sublineages (CAS, MANU2, EAI5, T2, LAM10_CAM, H1, X1, H4 and CAS2) involved altogether 24 (18.9%) isolates. Twenty-six (20.5%) isolates could not be assigned to any specific clade. MIRU-VNTR typing identified 123 (96.8%), 97 (76.4%) and 65 (51.2%) unique types with a tolerance of 0, 1, and 2 locus differences between the patterns.Upon combined spoligotyping and MIRU-VNTR typing analysis, 123 (96.8%), 108 (85%), and 91 (71.7%) unique types were identified if a tolerance of 0, 1, and 2 locus differences in the MIRU-VNTR patterns was assumed, respectively. Based on the clustering results, the transmission rate for MDR-TB cases under the study was calculated at 3.2%, 15%, and 28.3%.Overall, three clades, namely CAS1_DELHI, T1, and Beijing accounted for the majority of MDR-TB cases in Pakistan. Up to a third of the cases were clustered upon combined spoligotyping and MIRU-VNTR typing, suggesting a moderate level of active transmission.     

MIRU-VNTR和Spoligotyping用于重庆地区210株儿童结核分枝杆菌临床分离株的基因分型

目的 评价间隔区寡核苷酸分型(Spoligotyping)和基于结核分枝杆菌散在分布数目可变串联重复序列分析(MIRU-VNTR)方法在重庆地区儿童结核病分子流行病学中的应用.方法 收集重庆地区210株儿童结核分枝杆菌(MTB)临床分离株,应用上述两种分型方法进行比较分析.结果 采用Spoligotyping分型方法,210株菌可分为2个基因群44种基因型,其中最大的1个基因群即北京家族(北京基因型)含有130株菌(61.90%).采用MIRU-VNTR分析发现24个位点的多态性差异较大,不同MIRU位点组合(12、15和24位点)的分辨率指数依次升高,后两个组合的差异是由位点ETR-B引起.各位点和各位点组合在北京家族菌株中的分辨率指数均高于非北京家族菌株.结论 重庆地区儿童MTB具有明显的基因多态性,其主要流行型为北京家族.在结核病原学监测中,可先采用Spoligotyping,再对成簇菌株进行15位点与ETR-B组合二次分型的联合分型策略,可提高分子流行病学调查效果.

Abstract：

Objective To evaluate the application of spacer oligonucleotide typing (Spoligotyping) and mycobaeterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) analysis in the molecular-epidemiological study of tuberculosis and to discuss the characteristics of pediatric Mycobacterium (M.) tuberculosis strains in Chongqing. Methods M. tuberculosis strains isolated and typed by Spoligotyping and MIRU-VNTR respectively, from the children patients in Chongqing and to compare the results from both methods, epidemiologically. Results By means of Spoligotyping, 210 clinical isolates were divided into 2 gene groups, displaying 44 genotypes. Among them, the biggest group was M. tuberculosis Beijing family, including 130 strains (61.90%) ,using the Spoligotyping. From the results of MIRU-VNTR, 24 loci showed different polymorphism and the HGI of different loci set (12 old loci, 15 basic loci and 24-loci set) increased accordingly. The subtle difference in HGI was originated from one locus ETR-B, which was included in the 24-locus system. The diversity of each loci and MIRU-VNTR set for non-Beijing genotype strains was higher than that of the Beijing genotype strains. Conclusion In this study, it was preliminarily confirmed the existence of high polymorphism of M. tuberculosis while the Beijing Family was the main genotype and main prevalent strain in children of Chongqing area. Spoligotyping prior to 15-locus with ETR-B combination seemed more suitable for the massive epidemiological investigation of pediatric tuberculosis patients.     

Genetic diversity among multidrug-resistant Mycobacterium tuberculosis strains in Mexico

Tuberculosis is an important public health problem in Mexico. However, limited information about the genetic diversity of Mycobacterium tuberculosis strains circulating in the country is available. In this work, 109 multidrug-resistant (MDR) M. tuberculosis isolates collected in 23 different states of Mexico in 2003 were retrospectively characterized by spoligotyping and MIRU-VNTRs. All isolates, except for a single cluster containing two strains (subcluster E1), were split when information from the 12-loci MIRUs and spoligo-pattern was simultaneously analyzed. The discriminative power of 12-loci MIRU-VNTR and spoligotyping, by the Hunter-Gaston index, were 0.9998 and 0.9011, respectively. These findings suggest that almost all cases were epidemiologically unrelated. Instead, the genetic variations observed among these strains are suggestive of emergence of acquired drug-resistance during the course of treatment. The results suggest a high degree of genetic variability and a high frequency of SIT53 (T1 family) spoligotype among the MDR M. tuberculosis isolates included in the study.     

四川省结核分枝杆菌VNTR分型研究

目的评价不同串联重复序列(VNTR)位点在四川省结核分枝杆菌中的基因多态性,以及不同VNTR位点组合在四川省结核分枝杆菌基因分型中的应用。方法采用多位点数目可变串联重复序列分析技术(ML-VA)对102株四川省结核分枝杆菌菌株的15个VNTR位点进行分析,基因多态性分析采用Hunter-Gaston指数,聚类分析采用BioNumerics软件。结果 15个VNTR位点的基因多态性存在较大的差异,位点Mtub21(HGI 0.772)和MIRU26(HGI 0.764)的多态性较高,ETR-C(HGI 0.168)和MIRU23(HGI 0.077)的多态性较差。ETR-B和ETR-C两个位点在对四川省北京基因型结核分枝杆菌进行分型时则不具有多态性。对不同的VNTR位点组合进行比较,随着VNTR位点的增加,VNTR分型方法的分辨能力也有所提高。10个VNTR位点组合的分辨指数与15位点组合的分辨指数相差不大。同一VNTR位点在不同地区北京基因型菌株中的分辨力也存在较大的差异。结论不同VNTR位点在四川省结核分枝杆菌中具有不同的分辨力,并且同一VNTR位点在不同地区的北京家族菌株中的分辨力也不同。10个VNTR位点组合的基因分型方案可用于四川省结核分枝杆菌基因分型的一线方法。本研究的数据结果对挑选适合中国结核分枝杆菌基因分型的VNTR位点组合法具有重要的作用。     

Genotypes and genetic characters of Mycobacterium tuberculosis from Myanmar using three typing methods

Knowledge on basic characteristics of Mycobacterium tuberculosis (MTB) is helpful to understand the disease epidemiology and support the prediction of clinical outcome of the disease. The aim of this study was to detect the genotypes and genotypic characters of clinical Mycobacterium tuberculosis (MTB) isolates from new and retreatment rifampicin-resistant patients using three different genotyping methods. Mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) typing was used to determine the diversity of 222 clinical isolates. Spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) typing were also used to investigate the genetic characters of 105 MTB strains. Among the 15 genotypes detected by MIRU-VNTR, Beijing strains were the most prevalent of all strains (54.8%); new cases (40.5%) and retreatment cases (69.4%), followed by EAI strain. Spoligotyping categorized the strains into 11 lineages and 13 orphans whereas 96 different IS6110 patterns were identified using RFLP method. The mode number of IS6110 was 18 and 20. Higher band numbers were found in Beijing genotype (p < 0.001). Clustering rates by spoligotyping, MIRU-VNTR and IS6110-RFLP typing were 0.714, 0.004 and 0.085, respectively. Discriminatory powers of spoligotyping, MIRU-VNTR typing and IS6110-RFLP typing were 0.637, 1.000 and 0.997, respectively. Dominant Beijing genotype in both new and retreatment cases denoting that prevailing tuberculosis in Myanmar changed from EAI to Beijing lineage.     

Predominance of ancestral lineages of Mycobacterium tuberculosis in India

Although India has the highest prevalence of tuberculosis (TB) worldwide, the genetic diversity of Mycobacterium tuberculosis in India is largely unknown. A collection of 91 isolates originating from 12 different regions spread across the country were analyzed by genotyping using 21 loci with variable-number tandem repeats (VNTRs), by spoligotyping, by principal genetic grouping (PGG), and by deletion analysis of M. tuberculosis-specific deletion region 1. The isolates showed highly diverse VNTR genotypes. Nevertheless, highly congruent groupings identified by using the 4 independent sets of markers permitted a clear definition of 3 prevalent PGG1 lineages, which corresponded to the "ancestral" East African-Indian, the Delhi, and the Beijing/W genogroups. A few isolates from PGG2 lineages and a single representative of the presumably most recent PGG3 were identified. These observations suggest a predominance of ancestral M. tuberculosis genotypes in the Indian subcontinent, which supports the hypothesis that India is an ancient endemic focus of TB.     

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.     

Molecular clues of a microepidemy among homeless tuberculosis patients in Budapest due to a new and local Mycobacterium tuberculosis clade.

In Budapest, the capital of Hungary, one of the most important tuberculosis related risk factors is homelessness. The aim of this retrospective study was the genetic characterization of Mycobacterium tuberculosis strains isolated from 66 homeless tuberculosis patients by spoligotyping and mycobacterial interspersed repetitive unit (MIRU) typing. The study identified a local microepidemy in the district with the highest tuberculosis incidence of Budapest. Further genetic characterization has shown that the microepidemy was due to a locally emerged Budapest-specific lineage of M. tuberculosis. These data may serve as a reference to better monitor and understand the patterns and transmission dynamics of tuberculosis in this at-risk population and is the first report on genetic diversity of M. tuberculosis in today's Hungarians. The findings also indicate that tuberculosis control and prevention steps among the homeless need to be strengthened.     

Associations between Mycobacterium tuberculosis Strains and Phenotypes

To inform development of tuberculosis (TB) control strategies, we characterized a total of 2,261 Mycobacterium tuberculosis complex isolates by using multiple phenotypic and molecular markers, including polymorphisms in repetitive sequences (spoligotyping and variable-number tandem repeats [VNTRs]) and large sequence and single-nucleotide polymorphisms. The Beijing family was strongly associated with multidrug resistance (p = 0.0001), and VNTR allelic variants showed strong associations with spoligotyping families: >5 copies at exact tandem repeat (ETR) A, >2 at mycobacterial interspersed repetitive unit 24, and >3 at ETR-B associated with the East African–Indian and M. bovis strains. All M. tuberculosis isolates were differentiated into 4 major lineages, and a maximum parsimony tree was constructed suggesting a more complex phylogeny for M. africanum. These findings can be used as a model of pathogen global diversity.     

天津地区临床分离结核分枝杆菌分型的初步研究

目的探讨天津地区结核分枝杆菌临床分离株分子流行病学特征。方法连续收集天津市海河医院2005年8月16日11月25日就诊患者痰培养阳性的结核分枝杆菌100株，采用间隔区寡核苷酸分型（spoligotyping）和多位点可变串联重复序列（VNTR）两种方法进行基因分型，并运用软件对二者的结果进行分析。依据北京分化支的定义，运用多重和实时定量PCR方法将其区分为W菌／典型北京家族菌株和非典型北京菌株，Χ^2检验分析两种亚群与患者年龄和耐药性之间的联系。结果排除污染菌株，共对96株结核分枝杆菌临床分离株进行两种方法的基因分型，spoligotyping结果91．7％为北京基因型（含3株类北京基因型）结核分枝杆菌（88／96）。VNTR分型可将北京基因型分为60种基因型。在北京分化支结核分枝杆菌中，W菌／典型北京家族菌株占93．2％（82／88）。两种北京分化支亚群与患者年龄及耐药性之间差异无统计学意义（P〉0．05）。结论天津地区结核病患者临床分离的结核分枝杆菌中，北京基因型呈现较为明显的优势。VNTR的分辨率明显高于spoligotyping。北京分化支的两种亚群在天津地区临床结核病患者中均有流行，但以W菌／典型北京家族菌株为主。