华东农村地区利福平耐药结核病的 分子流行病学特征研究

目的 描述华东农村地区利福平耐药结核分枝杆菌(结核菌)的耐药相关分子特征和成簇性规律.方法 以浙江省和江苏省两个农业县结核病防治所一年内所有登记的结核菌检测阳性肺结核病例为研究对象,收集痰标本、培养分离菌株,并进行问卷调查;对培养获得的结核菌株采用比例法进行药敏试验.对获得的65株利福平耐药结核菌株进行rpoB基因利福平耐药决定区和katG基因异烟肼耐药相关热点区测序,采用间隔区寡核苷酸分型(spoligotyping)方法识别北京株,采用IS6110限制性内切酶多态指纹技术(RFLP)对分离株进行基因分型和成簇性分析.结果 92%(60/65)的利福平耐药菌株在利福平耐药决定区531位(57%)、526位(29%)或516位(11%)发生突变,82%(49/60)的rpoB基因突变菌株为耐多药结核菌(MDR-TB);spoligotyping识别H{54株(83%)北京家族菌株;IS6110-RFLP发现共有24株(37%)利福平耐药菌株成簇,形成11个簇,提示簇中病例的结核菌近期传播.所有成簇菌株都为MDR-TB.共有7个簇,簇内菌株具有不完全相同的利福平耐药相关的rpoB基因突变.分析可能与成簇性相关的因素,初治病例在菌株成簇患者中的比例高于在菌株非成簇患者中所占的比例(初治/复治:OR=3.342,95%CI:1.081～10.32).结论 在其他耐药结核菌(如异烟肼耐药结核菌)中进一步产生对利福平的选择性耐药和生长,很有可能是利福平耐药结核病流行的基础,而既往较长的不规则治疗史也为利福平耐药的发生和传播提供条件.     

结核分枝杆菌传播的分子流行病学特征

目的探讨结核分枝杆菌传播的基因型特征及其对于耐药结核菌近期传播的影响。方法选取13个社区作为监测点,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 558株结核分枝杆菌的12个MIRU位点检测产生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个菌株(31.7%)属于山东基因型;耐多药菌株的近期感染估计值(46.7%)明显低于全敏感菌株(72.7%)。结论山东基因型菌株具有较强的在人群中传播的能力、较高的耐药性以及对抗督导化疗的能力;利用基因分型技术开展结核病监测可以有效促进结核病控制工作。     

阿克苏地区耐药结核杆菌基因分型及耐药性分析

目的:初步了解新疆阿克苏地区耐药结核杆菌基因分型状况及影响耐多药产生的危险因素,为耐多药结核病防治提供有价值的信息。方法:收集阿克苏地区结核病专科医院(阿克苏第二人民医院)痰涂片阳性的维吾尔族肺结核病患者,从患者痰中分离并培养结核分枝杆菌并进行药物敏感试验,从186例临床分离株中筛选出65株耐药结核分枝杆菌,采用12个多位点数目可变串联重复序列(MIRU-VNTR)进行基因分型,同时应用logistic回归方法分析影响耐多药结核杆菌产生的危险因素。结果:65株耐药结核杆菌共产生52种基因型。其中独特基因型40种,余成12簇,每簇2～3株,共25株,成簇百分比38.5%(25/65),成簇率20%,耐多药成簇菌株12株,成簇百分比18.5%,成簇率11.5%。北京型耐多药菌株32株(61.5%),非北京型耐多药菌株20株(38.5%)。经单因素和多因素logistic分析显示,慢阻肺、复治、治疗中断或失败、肺空洞是MDR-TB产生的独立危险因素。结论:阿克苏地区耐药结核杆菌存在一定比例的近期传播,影响MDR-TB产生的危险因素有多种,需加强措施减少MDR-TB的产生和传播。     

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.     

用IS6110限制性片段长度多态性方法分析中国南方部队结核分支杆菌DNA指纹特征

目的：分析南方部队结核病患者和当地患者中结核分支杆菌分离株DNA指纹特征，探讨南方部队结核病的分子流行病学特征。方法：用限制性内切酶PvuⅡ消化结核分支杆菌DNA，后用琼脂糖凝胶电泳，再用Southern免疫转印，用[α^32P]-dCTP标记的DNA IS6110序列中的245bp片段作探针，进行杂交后得到限制性片段长度多态性图谱，结合一般流行病学资料加以分析比较。结果：共检测185株结核分支杆菌分离株。检测菌株的IS6110拷贝数范围为1～22。部队患者和当地患者的IS6110拷贝数分布差异无显著姓。部队患者结核菌分离株的IS6110拷贝数主要集中在6～20个，当地分离株主要集中在7～20个；全部菌株指纹特征分成8个组，部队分离株和当地分离株均主要集中在Ⅰ、Ⅱ、Ⅲ 3个组里。耐药菌株指纹特征在各组中的分布与敏感菌株差异有显著性；患者是否接种卡介苗在各组中的分布差异无显著性。结论：南方部队患者与当地患者结核菌分离株在遗传关系上较接近，在基因水平上相关程度较强。提示部队结核病的发生与当地结核分支杆菌菌株的传播密切相关。     

上海市静安区２０１０－２０１４年结核杆菌 耐药状况及传播特征研究

摘要：目的　了解上海市静安区结核病传播特征及结核分枝杆菌耐药情况,为制定有针对性的结核病控制 策略提供依据。方法　选择上海市疾病预防控制中心菌株库保存的２０１０－２０１４年静安区结核病患者菌株, 通过药物敏感性试验和可变数目串联重复序列分型,最后纳入７０ 株菌株进行耐药、ＶＮＴＲ 基因型分型结 果分析。结果　总耐药率为３０．００％ （２１／７０）,总耐多药率为１５．７１％ （１１／７０）,单耐药率为１２．８６％ （９／ ７０）,多耐药率为１．４３％ （１／７０）。无单耐利福平和单耐乙胺丁醇;初治与复治患者、男性与女性患者、＜ ６５岁与≥６５岁患者耐药率和耐多药率差异无统计学意义（犘＞０．０５）。７０ 株菌株共得到５７ 种ＶＮＴＲ 基因 型,其中５０例患者的分离株为单一基因型,其他２０例患者的菌株可归入７个基因簇,总成簇率为２８．５７％ （２０／７０）,同簇病例中有２簇存在家庭内接触,有５簇居住地间的直线距离在１．７ｋｍ 以内。耐多药菌株成 簇率高于全敏感结核菌株（χ ２＝９．４０６,犘＝０．００４）,差异有统计学意义。结论　上海市静安区结核分枝杆 菌的耐多药率较高,结核病患者中存在一定比例的近期传播,耐多药肺结核近期传播程度较高,高于全敏 感结核。 关键词：结核分枝杆菌;结核;耐药结核;基因型;耐多药 中图分类号：Ｒ３７８．９１１,Ｒ１８１．２　　文献标识码：Ａ　　文章编号：１００９ ６６３９ （２０１７）０９ ０６８０ ０５     

723例结核病住院患者结核分枝杆菌耐药性分析

目的 了解结核病住院患者结核分枝杆菌耐药情况.方法 收集2010年全年新收治结核病住院患者主要临床特征信息并进行统计分析,包括性别、年龄、糖尿病史、吸烟史及结核治疗史.回顾分析结核分枝杆菌临床分离株药敏试验结果.结果 2010年新分离分枝杆菌723株,其中结核复合群共671株,非结核分枝杆菌52株.671例结核病患者中,总耐药率43.82%、初治耐药率56.30%、复治耐药率32.07%;其中单耐药率、多耐药率和泛耐药率分别为13.41%、24.74%和5.66%.男女患者比例为2.2：1,患者年龄最小8岁,最大88岁.女性患者患耐药结核病风险低于男性,OR值为0.63（95%CI：0.42～0.94）,差异有统计意义（P〈0.05）.结核病治疗史和吸烟史是患者发生耐药结核的危险因素,OR值分别为1.62（95%CI：1.21～2.09）和2.72（95%CI：2.35～3.05）,差异有统计学意义（P均〈0.05）.非结核分枝杆菌株检出量及其检出率逐年上升,检出率由2006年的4.61%上升到2010年的7.19%.结论 应采取有效措施控制结核分枝杆菌感染及其耐药情况的进一步恶化.     

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。     

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。     

某区2004年中、小学生结核病普查

目的：评估本地区中小学生结核病流行病学状况。方法：用传统流行病学和分子流行病学方法。对125893名中、小学生进行PPD实验和X线检查；结核病人进行痰结核菌涂片和培养；对结核分枝杆菌分离株进行分子流行病学研究，用标准RFLP技术使用IS6110插入序列对结核分枝杆菌进行分型和成簇分析。结果：125893名中、小学生中（95．9％）接种过BCG。小学生1～4年级PPD阳性20963例，阳性率47．9％，其中强阳性17例；确定诊断结核感染3例、颈淋巴结结核4例，原发综合征和肺门淋巴结结核10例。小学5、6年级和中学生共X线异常阴影并确诊为肺结核48例。58例肺结核病人痰抗酸染色阳性15例（25．9％）、培养阳性42例（72．4％）；患病率46．1／10万；涂阳患病率13．5／10万。42株结核分枝杆菌中，IS6110 RFLP分析23株（55％）成簇，拷贝相似性高；其余19株IS6110拷贝呈现多态性。最大簇包含3例病人。结论：本地区中、小学生结核病患病率较低；新近传播病例未形成较大成簇，近期可能无集团感染和暴发发生。     

2014 - 2017年新余市结核病耐药特征及影响因素分析

目的 了解新余市耐药结核病的流行特征及其影响因素,为耐药结核病防治和管理提供科学依据。方法 2014 - 2017年新余市所有涂阳肺结核病人痰标本进行培养,用比例法进行药敏试验,用SPSS 19.0进行数据分析。结果 新余市2014 - 2017年涂阳结核病标本340例,其中6.47%（22例）为非结核分枝杆菌,93.53%（318例）为结核分枝杆菌。结核分枝杆菌的总耐药率20.44%（65例）,单耐药率6.60%（21例）,多耐药率4.09%（13例）,耐多药率9.75%（31例）。初治病人耐药率15.44%（42/272）,复治病人耐药率50.00%（23/46）。年龄(OR = 1.023,95%CI = 1.006～1.040) 、吸烟史(OR = 2.126,95%CI = 1.111～4.067)、居住农村(OR = 0.468,95%CI = 0.233～0.938)和抗结核病治疗史(OR = 4.580,95%CI = 2.291～9.158)是新余市结核病发生耐药的主要影响因素。结论 新余市结核病耐药形势严峻,结核病耐药率和耐多药率均显著高于世界平均水平,耐多药结核菌株中以同时耐6种药物为主。应提高初治病人的治愈率和加强耐药结核病防控工作。     

海南省结核分枝杆菌耐药影响因素分析

目的探讨海南省结核菌耐药影响因素,为制定耐药结核病防控措施提供科学依据。方法将全省18个市县均纳入调查点,通过调查问卷获取患者相关信息,采用比例法进行药敏试验,检测药物包括异烟肼(H)、利福平(R)、链霉素(S)、乙胺丁醇(E)、卡那霉素(Km)和氧氟沙星(Ofx);采用多因素非条件Logistic回归分析耐药的影响因素。结果纳入耐药分析的患者1 155例,其中初治880例,复治275例。总体耐药率和耐多药率分别为20.87%、7.53%。影响因素分析表明,对于初治患者,女性(χ2=4.35,P=0.04,OR=0.51,95%CI=0.27~0.96)和年龄的增长(χ2=4.03,P=0.04,OR=1.02,95%CI=1.00~1.04)是发生耐药、耐多药结核病的危险因素;对于复治患者,接受过2次以上抗结核药物治疗(χ2=4.07,P=0.04,OR=0.61,95%CI=0.38~0.99)、首次在综合医院治疗(χ2=5.28,P=0.02,OR=3.06,95%CI=1.18~7.96)、无人监督服药(χ2=4.99,P=0.03,OR=0.43,95%CI=0.21~0.90)、有药物副反应(χ2=5.71,P=0.02,OR=2.42,95%CI=1.17~4.98)发生耐药的风险较高,而产生耐多药的危险因素主要是有药物副反应发生(χ2=10.14,P=0.00,OR=3.40,95%CI=1.60~7.23)。结论女性及年老的初治患者发生耐药、耐多药结核病的风险较高;患者初次治疗在综合医院、有药物副反应发生、无人监督服药且多次治疗是复治患者产生耐药的危险因素。     

Genotyping analyses of tuberculosis cases in U.S.- and foreign-born Massachusetts residents

We used molecular genotyping to further understand the epidemiology and transmission patterns of tuberculosis (TB) in Massachusetts. The study population included 983 TB patients whose cases were verified by the Massachusetts Department of Public Health between July 1, 1996, and December 31, 2000, and for whom genotyping results and information on country of origin were available. Two hundred seventy-two (28%) of TB patients were in genetic clusters, and isolates from U.S-born were twice as likely to cluster as those of foreign-born (odds ratio [OR] 2.29, 95% confidence interval [CI] 1.69 to 3.12). Our results suggest that restriction fragment length polymorphism analysis has limited capacity to differentiate TB strains when the isolate contains six or fewer copies of IS6110, even with spoligotyping. Clusters of TB patients with more than six copies of IS6110 were more likely to have epidemiologic connections than were clusters of TB patients with isolates with few copies of IS6110 (OR 8.01, 95%; CI 3.45 to 18.93).     

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.     

IS6110 Restriction Fragment Length Polymorphism Typing of Drug-resistant Mycobacterium tuberculosis Strains from Northeast South Africa

Tuberculosis (TB) remains a deadly infectious disease affecting millions of people worldwide; 95% of TB cases, with 98% of death occur in developing countries. The situation in South Africa merits special attention. A total of 21,913 sputum specimens of suspected TB patients from three provinces of South Africa routinely submitted to the TB laboratory of Dr. George Mukhari (DGM) Hospital were assayed for Mycobacterium tuberculosis (MTB) growth and antibiotic susceptibility. The genetic diversity of 338 resistant strains were also studied. DNA isolated from the strains were restricted with Pvu II, transferred on to a nylon membrane and hybridized with a PCR-amplified horseradish peroxidase 245 bp IS6110 probe. Of the 338 resistant strains, 2.09% had less than 5 bands of IS6110, and 98% had 5 or more bands. Unique restriction fragment length polymorphism (RFLP) patterns were observed in 84.3% of the strains, showing their epidemiological independence, and 15.7% were grouped into 22 clusters. Thirty-two strains (61.5%) from the 52 that clustered were from Mpumalanga, 16/52 (30.8%) from Gauteng, and 4/52 (9.6%) from Limpopo province. Clustering was not associated with age. However, strains from male patients in Mpumalanga were more likely to be clustered than strains from male patients in Limpopo and/or Gauteng province. The minimum estimate for the proportion of resistant TB that was due to transmission is 9.06% (52-22=30/331). Our results indicate that transmission of drug-resistant strains may contribute substantially to the emergence of drug-resistant tuberculosis in South Africa.Key words: Drug resistance, Epidemiology, IS6110, M. tuberculosis, PCR-RFLP, South Africa     

Worldwide emergence of extensively drug-resistant tuberculosis

Mycobacterium tuberculosis strains that are resistant to an increasing number of second-line drugs used to treat multidrug-resistant tuberculosis (MDR TB) are becoming a threat to public health worldwide. We surveyed the Network of Supranational Reference Laboratories for M. tuberculosis isolates that were resistant to second-line anti-TB drugs during 2000-2004. We defined extensively drug-resistant TB (XDR TB) as MDR TB with further resistance to > or = 3 of the 6 classes of second-line drugs. Of 23 eligible laboratories, 14 (61%) contributed data on 17,690 isolates, which reflected drug susceptibility results from 48 countries. Of 3,520 (19.9%) MDR TB isolates, 347 (9.9%) met criteria for XDR TB. Further investigation of population-based trends and expanded efforts to prevent drug resistance and effectively treat patients with MDR TB are crucial for protection of public health and control of TB.     

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.