220株结核分枝杆菌北京临床分离株的基因分型研究

目的了解结核分枝杆菌北京临床分离株的基因分型种类和特征,为结核病防治研究提供基础科学依据。评价两种分型方法在北京地区结核病分子流行病学中的应用。方法收集北京市结核分枝杆菌临床分离株,应用间隔区寡核苷酸分型(Spoligotyping)及多位点可变数目串联重复序列分析(MLVA)二种分型方法进行基因分型,结果聚类分析采用BioNu-merics(Version5.0)软件。结果共在北京地区收集到220株结核分枝杆菌临床分离株。采用Spoligotyping分型方法,220株菌可分为2个基因群,即北京家族(Beijing family)和非北京家族(non-Beijing family),20种基因型。其中北京家族含有202株菌,占91.82%。北京家族菌株中,典型北京家族菌株占95.05%(192/202),非典型北京家族占4.95%(10/202)。北京家族菌株的耐药率为22.55%(22/98),非北京家族菌株的耐药率为16.67%(1/6),两者间的差异无统计学意义(fisher analysis,P=0.5835>0.05)。采用MLVA分型方法,202株菌可分成5个基因群59种基因型,主要为Beijing family、China2和China3,分别占91.37%、2.73%和4.55%。结论北京地区结核分枝杆菌具有明显的基因多态性,其主要流行型为北京家族。北京家族菌株与耐药性无明显相关性。     

青海省251株结核分枝杆菌Spoligotyping基因型与4种一线药物耐药表型的研究

目的研究青海结核分枝杆菌对4种一线抗结核药物耐药状况及与Spoligotyping基因型的关系,为结核病有效预防提供依据。方法采用常规比例法对分离的251株结核分枝杆菌进行异烟肼(INH)、利福平(RFP)、链霉素(SM)和乙胺丁醇(EMB)等4种一线抗结核药物的药敏试验,并对菌株进行Spoligotyping分型,对药敏试验和基因分型结果进行综合分析。结果 251株结核分枝杆菌中,总耐药率为56.2%;对INH、RFP、SM和EMB的耐药率分别为43.0%(108/251)、37.1%(93/251)、39.0%(98/251)和27.9%(70/251);耐多药率为31.5%(79/251)。所有菌株经Spoligotyping分型,分为北京基因型185株(73.7%)与非北京型菌株66株(26.3%)。未发现北京型与非北京型结核菌株与耐药存在统计学关联。结论青海流行的结核分枝杆菌耐药率及多耐药率较高,Spoligotyping分型显示北京基因型为主要流行型。     

耐异烟肼结核分枝杆菌临床分离株耐药基因突变及其分子流行病学调查研究

目的阐明结核分枝杆菌耐异烟肼临床分离株katG基因突变特点以及Spoligotyping分型和结核病流行关系。方法对123株结核分枝杆菌临床分离株，其中有药敏结果的98株，（耐异烟肼菌株45株；异烟肼敏感株53株）的katG基因进行DNA片断扩增然后进行SSCP分析；同时对其中的121株菌株进行Spoligotyping分型。结果45株耐INH结核杆菌株中，27株（60％）第315位点突变，低耐药菌株（1mg／L）第315位点突变率显著高于高耐药菌株（10mg／L；53株敏感株中无KatG基因315位密码子突变。对121株临床株的Spoligotyping DNA指纹分析，1型，（北京型）103株占84％（103／121），其它基因型18株，分散在13种基因型中。结论本项研究进一步证实了结核分枝杆菌耐异烟肼与katG基因突变之间的关系；北京型感染是北京地区结核病流行的重要原因。     

重庆市结核分枝杆菌临床分离株的基因分型及相关耐药性分析

目的 对重庆市结核分枝杆菌临床分离株进行基因分型,研究不同基因型在重庆的流行情况,并分析基因型与耐药表型的关系。 方法 收集2011年1-12月重庆市结核病防治所分离的113株结核分枝杆菌临床分离株,采用比例法进行药物敏感性检测;应用间隔区寡核苷酸分型（Spoligotyping）和PCR方法对113株结核分枝杆菌临床分离株进行基因分型。 结果 在113株结核分枝杆菌临床分离株中,可分为北京家族和非北京家族,分别占77株（68.1%,77/113）和36株（31.9%,36/113）;北京家族菌株中现代型为48株（62.3%,48/77）,古代型为29株（37.7%,29/77）;非北京家族菌株中,T1型高达13株（36.1%,13/36）,发现9种新基因型。北京家族菌株耐药率为24.7%（19/77）,非北京家族菌株耐药率为38.9%（14/36）,差异无统计学意义（χ²=2.40,P>0.05）。现代型北京家族菌株耐药率为27.1%（13/48）,古代型北京家族菌株耐药率为20.7%（6/29）,差异无统计学意义（χ²=0.39,P>0.05）。 结论 重庆地区结核分枝杆菌具有明显的基因多态性,其主要流行基因型为北京家族,其中北京家族中主要为现代型;北京家族菌株、非北京家族菌株均与耐药性无明显相关性。     

结核病分子流行病学研究方法进展和探讨

结核病分子流行病学在结核病的控制方面有着良好的应用前景,发达国家已将其列入常规检查,在我国也有很好的应用前景和更大的应用空间.目前的基因分型的主流方法仍然是采用数目可变串联重复序列(VNTR)和间隔区寡核苷酸分型共同应用的模式.间隔区寡核苷酸分型是继IS6110限制性片段长度多态性(RFLP)之后应用于结核分枝杆菌基因分型最广泛的方法之一,目前被作为鉴定北京基因型菌株的标准方法.VNTR方法操作简便、快速,成本低廉,重复性好,便于不同实验室间相互比较;VNTR方法选用的位点在不同国家和地区会有不同,VNTR位点分辨率的高低也需要深入研究.IS6110-RFLP方法虽然已经不再大规模地使用,但是一直作为鉴定其他基因分型方法的正确率和准确度的金标准.随着分子生物学技术的进步,基因分型技术出现新的发展趋势.如长片段多态性(LSP)和单核苷酸多态性(SNP)方法是近几年出现的频率越来越高的分型方法.目前LSP和SNP方法的分辨率虽然不高,但在研究系统发育中确有很好的应用.系统发育学的研究一开始是采用VNTR-15和VNTR-24的方法,后来发现存在不确定性且误差较大,随着对LSP和SNP特性的了解更加全面,认为LSP和SNP是研究系统发育学的最好的方法.随着分子生物学技术的不断进步,结核病分子流行病学的研究将会为结核病的预防和控制提供更多支持和指导.     

Spoligotyping对广西地区208株结核分枝杆菌临床分离株的基因分型

目的用间隔区寡核苷酸分型(Spoligotyping)对广西地区结核分枝杆菌临床分离株进行基因分型,并分析北京家族菌株与耐药的相关性。方法收集广西地区结核分枝杆菌临床分离株,应用Spoligotyping进行基因分型研究和比例法进行药物敏感性检测。基因聚类分析采用BioNumerics软件。统计分析采用χ2检验。结果共在广西地区收集到208株结核分枝杆菌临床分离株,可分为2个基因群,即北京家族(Beijingfamily)或称北京基因型(Beijinggenotype),和非北京家族(Non-Beijingfamily),分别占55.3%(115/208)和44.7%(93/208),47种基因型,其中36株为独特类型,剩余172株分为11簇。北京家族菌株中,90.4%(104/115)为典型北京家族(TypicalBeijingfamily),非北京家族菌株表现为高度的基因多态性,可分为39个基因型,31株为独特的基因型。有耐药结果的205株菌株中,北京家族菌株,76.25%(76/113)表现为全敏感,而23.75%(37/113)表现为耐药;非北京家族菌株,75%表现为全敏感,而25%表现耐药,经卡方检验(Chi-SquareTests)(表2),两者间的差异无统计学意义(χ2=2.9721,P>0.05)。结论广西结核分枝杆菌存在明显的基因多态性。本研究证实北京基因型与耐药无明显相关性。     

结核分枝杆菌对氨基水杨酸耐药的分子机制及研究进展

结核病由结核分枝杆菌感染引起,是我国面临的重大传染病,严重危害人民健康。而近年来日益增长的耐药结核病疫情使我国结核病防控工作面临严峻挑战。对氨基水杨酸(PAS)是治疗耐多药结核病的重要药物,其机制是抑制结核分枝杆菌叶酸的合成。随着其广泛运用,目前部分临床菌株已形成对PAS的耐药性。结核分枝杆菌叶酸代谢通路相关基因fol C、thy A和rib D突变导致对PAS耐药的分子机制已较为明确。此外,近期有研究揭示了一些新的PAS潜在作用靶标及耐药位点。本文综述了PAS作用机制及结核分枝杆菌对PAS耐药机制的研究进展,旨在为研究新的PAS耐药机制提供参考。     

结核分枝杆菌耐药性检测方法的研究进展

结核分枝杆菌耐药基因的检测是结核病诊断和治疗中的难题之一,近年来已有多种方法用来检测耐药基因,笔者从在我国获得注册证书的试剂盒,在国际上获得注册证书尚未进入我国的试剂盒和临床前研究的新方法3个方面概述了结核分枝杆菌耐药性检测方法的研究进展。     

复发肺结核患者再感染病原学研究带给我们的启示

本期发表的梅建等《原发性耐药是耐药结核病产生的重要原因》和沈国妙等《利用结核分枝杆菌基因型分型技术研究外源性再感染在结核病复发中的作用》二文，会令我们对肺结核复发患者再感染的病原学产生新的认识。     

结核分枝杆菌耐氯法齐明相关基因研究进展

目前,全球范围内结核病防控形势仍十分严峻,且耐药结核病所占比例较高,因此不断有新型抗结核病药物被研发出来并应用于临床。氯法齐明是目前世界卫生组织(WHO)推荐的二线核心抗结核药物之一,主要用于治疗耐多药结核病(MDR-TB)且疗效肯定,但近年来结核分枝杆菌对氯法齐明耐药现象增多。本文综述了结核分枝杆菌耐氯法齐明相关基因,旨在为结核分枝杆菌对氯法齐明的耐药机制研究提供参考。     

The Ontario Universal Typing of Tuberculosis (OUT-TB) Surveillance Program �C what it means to you

BACKGROUND:

Tuberculosis (TB) is a serious disease that is transmitted primarily by the airborne route. Effective disease control and outbreak management requires the timely diagnosis, isolation and treatment of infected individuals with active disease; contact tracing to identify secondary cases likely to benefit from treatment of latent infection; and laboratory identification or confirmation of epidemiologically linked cases. TB genotyping enables the comparison of Mycobacterium tuberculosis complex (MTBC) strains and the identification of cases that may or may not be linked. The increased availability of molecular methods for genotyping has allowed for greater discrimination of MTBC strains and greatly enhanced understanding of TB transmission patterns.

OBJECTIVE:

To improve TB surveillance and control in Ontario, the Public Health Laboratories of the Ontario Agency for Health Protection and Promotion has introduced the Ontario Universal Typing of Tuberculosis (OUT-TB) Surveillance Program.

METHODS:

The first isolate from every new TB case will be genotyped with two rapid molecular methods: spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing. MTBC isolates with nonunique genotypes and, thus, potentially linked to other TB cases, will also be genotyped by IS6110 restriction fragment length polymorphism analysis.

CONCLUSION:

By providing TB control programs using these new genotyping tools, and using traditional and new case investigation methods (eg, social network analysis), this new program will provide a clearer picture of TB in Ontario, and permit more effective use of public health resources and improve disease control.     

Transmission network analysis in tuberculosis contact investigations

BACKGROUND: Social network analysis (SNA) is an innovative approach to the collection and analysis of infectious disease transmission data. We studied whether this approach can detect patterns of Mycobacterium tuberculosis transmission and play a helpful role in the complex process of prioritizing tuberculosis (TB) contact investigations. METHODS: We abstracted routine demographic and clinical variables from patient medical records and contact interview forms. We also administered a structured questionnaire about places of social aggregation to TB patients and their contacts. All case-contact, contact-contact, case-place, and contact-place dyads (pairs and links) were considered in order to analyze the structure of a social network of TB transmission. Molecular genotyping was used to confirm SNA-detected clusters of TB. RESULTS: TB patients not linked through conventional contact-investigation data were connected through mutual contacts or places of social aggregation, using SNA methods. In some instances, SNA detected connected groups prior to the availability of genotyping results. A positive correlation between positive results of contacts' tuberculin skin test (TST) and location in denser portions of the person-place network was observed (P<.01). CONCLUSIONS: Correlation between TST-positive status and dense subgroup occurrence supports the value of collecting place data to help prioritize TB contact investigations. TB controllers should consider developing social network analysis capacity to facilitate the systematic collection, analysis, and interpretation of contact-investigation data.     

Tuberculosis in the Inuit community of Quebec, Canada

In low-incidence countries targeting tuberculosis (TB) elimination, TB remains a problem of a few high-risk groups. In Canada, Aboriginals, and particularly the Arctic Inuit communities, have witnessed dramatic decreases in TB during the 1960s to 1970s, but rates remain at least 10 to 20 times higher than the national average. We are describing the results of an integrated traditional and molecular epidemiology study of all culture-positive Mycobacterium tuberculosis cases in the Arctic Inuit communities of Quebec from 1990 until 2000. The demographic characteristics of the 46 TB cases included in the study were most notable for a bimodal age distribution (48% under 25 years). Genotyping analysis using multiple modalities (IS6110 restriction fragment length polymorphism, spoligotype, mycobacterial interspersed repetitive units-variable number tandem repeats) showed that 76% (35/46) of TB cases were clustered (six clusters, median size four cases) and estimated that at least 62.5% of TB cases were due to ongoing transmission. By integrating the epidemiologic and genotyping data, we observed that the genotyping clustering results were concordant with recognized epidemiologic links but most notably identified previously unrecognized intervillage transmission. This study demonstrates significant ongoing transmission in a geographically isolated, low-density population. In a resource-rich country such as Canada, these communities illustrate some of the persistent challenges of TB control and elimination.     

复发与治疗失败患者结核分枝杆菌基因型分析

目的分析复发和治疗失败患者结核杆菌基因型并进行病原学探讨。方法以2000年1月—2007年12月在北京市结核病胸部肿瘤研究所2次住院相邻时间间隔至少在6个月以上、有分离菌株的肺结核病患者作为研究对象。应用结核分枝杆菌基因分型方法对患者初次和第2次治疗的菌株进行分型并比较。结果24例结核病患者入选本研究。在完成初次治疗的5人中,其前后分离株的基因型均相同;在另19例患者（8例丢失、11例治疗失败）中,有2例(10.5%)患者其分离株的基因型与第1次不同;其余17例（89.5%）患者前后2次分离株的基因型相同。结论内源发病是复发的主要原因,失败和丢失病例中少数存在再感染情况。     

Recommendations on modern contact investigation methods for enhancing tuberculosis control

Effective contact investigations are paramount to the success of tuberculosis (TB) control in high-risk communities in low TB prevalence countries. National and international guidelines on TB contact investigations are available and vary widely on recommendations. Because of the limitations of traditional contact tracing, new approaches are under investigation, and in some cases in use, to ensure effective TB control in those persons and communities at greatest risk. These non-traditional approaches include the use of social network analysis, geographic information systems and genomics, in addition to the widespread use of genotyping, to better understand TB transmission. Detailed guidelines for the use of these methods during TB outbreaks and in routine follow-up of TB contact investigations do not currently exist despite evidence that they may improve TB control efforts. It remains unclear as to when it is most appropriate and effective to use a network-informed approach alone, or in combination with other methodologies as well as the extent of data collection required to inform practice. TB controllers should consider developing the capacity to facilitate the systematic collection, analysis, and interpretation of contact investigation data using such novel methodologies, particularly in high-risk communities. Further investigation should focus on questionnaire development and adaptation, electronic data management and infrastructure, development of local capability and consultant expertise, and the use of coordinated approaches, including deployment strategies and evaluation.     

Molecular epidemiology of Mycobacterium tuberculosis and its prospect based on variable number of tandem repeat (VNTR) genotyping--a strategy in Osaka City, Japan

The methodological establishment of variable number of tandem repeat(s) (VNTR) genotyping of Mycobacterium tuberculosis has opened a new era of molecular epidemiology against tuberculosis (TB). The method can provide simple, rapid and accurate identification of clinical isolates from TB patients that makes it possible to compare the isolates among different laboratories. Such advantages of VNTR not only help us certify the identification of isolates in putative outbreaks easily but also promote the reasonable estimation of unidentified transmissions in surveillance studies. Recently, the Japan Anti-Tuberculosis Association (JATA) (12)-VNTR has become a standard genotyping method of M. tuberculosis, and its spread has been expected in Japan. In Osaka City, located in the western part of the country, JATA (12)-VNTR has been applied to molecular epidemiological study of TB. Moreover, the additional 12 VNTR loci have been analyzed for various purposes, such as to enhance the discriminatory power (public health needs) or to further analyze the population genetic structure (research needs). As the nationwide findings of VNTR genotyping of M. tuberculosis are accumulated, this technology will be increasingly useful for detecting transmission of any specific strain in large geographic areas that could not be recognized by conventional epidemiological methods. The needs for the VNTR genotyping of M. tuberculosis and its practical uses are expected to expand drastically in the future.     

Transmission of Mycobacterium tuberculosis to a funeral director during routine embalming

Several studies have shown that funeral directors have an increased risk of tuberculosis (TB). Although there is indirect evidence of transmission of TB from cadavers to mortuary workers, there is only one recently documented case in the literature. We report here another case of occupationally acquired TB in a funeral director, which was confirmed by conventional epidemiology and genotyping. This case illustrates the risk of TB transmission to mortuary workers from routine embalming of deceased TB patients with active disease.     

采用分子流行病学和传统调查相结合分析嘉祥县某中学结核病聚集性疫情

目的 分析2007-2008年山东省济宁市嘉祥县某中学一起结核病聚集性疫情的情况。 方法 以线索病例为依据,对该校3254名学生进行PPD试验和症状筛查。对分离培养的Mtb进行24位点分枝杆菌散在重复单位-可变数目串联重复序列(Mycobacterial interspersed repetitive unit-variable number tandem repeat,MIRU-VNTR)基因分型分析。采用logistic回归分析法,分析菌阳患者暴露程度和确诊为活动性结核病患者（active tuberculosis,ATB）或潜伏感染者(LTBI)的关系。 结果 筛查该中学学生的Mtb潜伏感染率为28.8%,共确诊10例活动性肺结核患者,其中3例培养阳性。24位点MIRU-VNTR基因分型3株菌株结果显示,2株菌株基因型成簇,1株菌株位点Mutub4、Mtub21和QUB-26拷贝数不同。logistic回归分析结果显示与菌阳患者同班级或同年级是被诊断为ATB或LTBI的危险因素。 结论 该次结核疫情中,3位菌阳患者在学校引起了一定程度的传播,可能导致了另外7例ATB的暴发。因此对学生结核病患者的及时确诊和有效治疗,以及对学生及时进行结核病筛查对控制学校结核病暴发具有重要意义。     

结核病患者N-乙酰基转移酶2基因型与异烟肼血药浓度关系的研究

目的 探讨结核病患者N-乙酰基转移酶2(NAT2)基因型与异烟肼(INH)血药浓度的关联性,为临床根据NAT2基因分型指导合理INH用药提供依据。 方法 应用PCR结合限制性片段长度多态性（PCR-RFLP）方法对121例住院结核病患者NAT2基因型分布进行检测,同时应用液相色谱-串联质谱分析仪（LC/MS-MS）检测结核病患者服药2h后血浆INH浓度。所获数据采用单因素方差分析检测组间差异,进而采用Tamhane’s T2法进行两两比较,以P＜0.05为差异有统计学意义。 结果121例结核病患者NAT2基因型中快速乙酰化型（RA,野生型）有55例,INH血药浓度为（1.86±1.28）μg/ml;慢乙酰化型（SA,纯合突变型）有17例,INH血药浓度为（5.86±2.10）μg/ml;中间乙酰化型（IA,杂合突变型）有49例,INH血药浓度（3.49±2.60）μg/ml。住院结核病患者整体INH血药浓度均值为（3.08±2.42）μg/ml。三型INH血药浓度比较,差异均具有显著统计学意义（RA与IA,P＝0.001;RA与SA,P＝0.002;IA与SA,P＝0.000）。结论 不同NAT2基因型人群对INH的代谢能力差异存在显著统计学意义,NAT2基因型分析对结核病患者INH用药具有重要指导意义。     

New era in molecular epidemiology of tuberculosis in Japan

Molecular epidemiology of tuberculosis (TB) is a science to study TB transmission dynamics and to enhance our understanding of the epidemiology of TB by utilizing molecular typing methods as an adjunct to classical epidemiological approach. Before the era of molecular epidemiology, it was quite difficult to ascertain the source of the infections since M. tuberculosis is spread by air-borne droplets of respiratory secretions expelled by an infectious person to a susceptible host and it can remain latent as an asymptomatic infection for years. Now a day, our understanding of TB transmission dynamics has been refined by genotyping of M. tuberculosis strains. The methods of molecular epidemiology, especially IS6110 RFLP of M. tuberculosis, were first introduced to outbreak investigations and then gradually been expanded its application to population-based study in Japan. IS6110 RFLP is obviously a powerful tool for strain differentiation of M. tuberculosis but its labor-intensiveness limits the achievable throughput and makes it less useful for long-term prospective studies. Recently, apart from IS6110 RFLP, DNA amplification-based method, i.e., variable number of tandem repeats (VNTR) has appeared as a substitute for or adjunct to the IS6110 RFLP. In this symposium, we have invited four opinion leaders in molecular epidemiology of TB from different fields: Mycobacterium reference center, basic science, clinical practice, and public health practice. We, as the chairpersons of this symposium, hope that this symposium would trigger the development of molecular epidemiological network of TB in Japan. 1. Achievement and problem of molecular epidemiologic study with IS6110-RFLP analyses of tuberculosis in Okinawa: Shinji MAEDA (Mycobacterium Reference Center, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association) The long-term RFLP analyses of tuberculosis in Okinawa showed that endemic M. tuberculosis might be present. This is one of the achievements of our project study. On the other hand, for more effective examination of contact persons, information of molecular epidemiology should be used actively. Therefore because the analysis report needs to be sent back quickly, the PCR-based VNTR method should substitute for the RFLP analysis. 2. Basic knowledge and application of Variable Numbers of Tandem Repeats: Kei NISHIMORI (Department of epidemiology, National Institute of Animal Health) Genomic loci of Variable Numbers of Tandem Repeats (VNTR loci) in Mycobacterium tuberculosis complex and Mycobacterium avium, the history of analysis of VNTR loci, the hypothetical mechanisms of increase or decrease of number of repeats, the structures of the loci, and the necessity of standardizing the VNTR typing were introduced. 3. Clinical application of VNTR: Tomoshige MATSUMOTO, and Hiromi ANO (Department of Clinical Research and Development, Osaka Prefectural Hospital Organization Osaka Prefectural Medical Center for Respiratory and Allergic Diseases) Tuberculosis genotyping was first introduced to outbreak investigations and population-based studies. The advent of Variable Numbers of Tandem Repeats (VNTR) can be applied to clinical fields of not only Mycobacterium tuberculosis but also of Mycobacterium avium. In Osaka Prefectural Medical Center for Respiratory and Allergic Diseases, clinical application of VNTR was first introduced in Japan to determine whether Mycobacterium tuberculosis or avium disease was caused by reactivation or reinfection when relapsed. We showed some examples about usefulness of the clinical application of VNTR. 4. Molecular epidemiology of tuberculosis to improve TB prevention and control activities: Tomotada IWAMOTO (Department of microbiology, Kobe Institute of Health), Riyo FUJIYAMA, Noriko TANAKA, Yasuto KAWAKAMI (Kobe City Public Health Center), Chika SHIRAI (Hyogo-ku Health and Welfare Department, Kobe) M. tuberculosis isolates in Kobe have been characterized as: a) Beijing family strains are highly prevalent (77%), b) two major MIRU profiles in Beijing family were found, one is globally pandemic genotype and the other is locally prevalent strains, c) six strains belonged to T3-Osaka family, and d) Manila family strains made cluster consisting of 3 strains. Kobe VNTR Database which consists of 12-loci MIRU and 9 additional VNTR loci has been developed. The basis for the selection of these supplemental 9 VNTR loci and the application of VNTR database in TB control program were introduced.