【作者】 胡屹；

【导师】 徐飚；

【作者基本信息】 复旦大学 ， 流行病与卫生统计学， 2008， 博士

【摘要】 我国早在上世纪90年代就开始实施以直接督导下的短程化疗(Directlyobserved treatment,short course,DOTS)策略为基础的国家结核病控制项目。到目前为止,已经免费治疗了大约一千三百万传染性肺结核病人,治疗成功率高达94%以上,基本完成了世界卫生组织制定的治疗和发现结核病病人的目标。但是就结核病的疾病负担(病例数)而言,中国仍然是全球结核病第二大高负担国家。我国拥有世界上16%的结核病人,2004年我国的涂阳结核病和活动性结核病报告率分别为46/100,000和101/100,000。近几年来,中国结核病控制面临的一个更为严峻的问题是耐药(尤其是耐多药)结核病的流行。耐多药结核病是指结核病患者感染的结核杆菌体外被证实至少对异烟肼、利福平这两种抗结核一线药物耐药。目前全球新发结核病病人中的耐多药结核病率(Multidrug-resistant tuberculosis,MDR-TB)平均为2.7%,有即往结核病治疗史的病人中MDR-TB发生率为4.3%。而我国2004年报告的MDR-TB发生率高于全球平均水平,初治病人为5.3%,复治病人为27.2%。全国第四次结核病流调发现,结核病病人中的初始总耐药率为18.7%,MDR-TB率为6%。不同来源的监测数据和研究结果显示,我国部分地区已成为全球耐药结核病的高流行区,耐药结核菌株的传播和流行已经成为影响中国结核病控制项目顺利实施的主要障碍之一。MDR-TB可以直接导致结核病的高患病率、高病死率,并可造成治疗过程延长和治疗成本增加,还可在社区人群中传播引起MDR-TB的暴发流行。本研究以江苏省和浙江省农村两个实施DOTS策略的结核病控制项目县为研究现场,采用现场流行病学与现代分子生物学技术相结合的方法,对两县研究期间的初治和复治结核病病人进行了系统的研究。本研究运用横断面调查方法,描述耐药结核病的流行现状、耐药结核病类型及其在不同人口学和社会经济水平人群中的分布,分析影响农村地区耐药结核病流行的生物、人口学和社会经济因素。并在现场流行病学研究的基础上,运用MIRU,Spoligotyping,IS6110等基因分型技术对收集的结核分枝杆菌(mycobacteria tuberculosis,M.TB)进行基因分型,确定其所属细菌家族,采用成簇性分析比较不同耐药类型分离株中近期感染和复燃比例:利用直接测序法,描述所有与一线抗结核药物耐药有关的已知基因突变热点区、基因突变型及其与耐药表型的关系;借助社会学和流行病学调查手段,深入探讨耐药结核病在农村的传播模式,分析北京家族及其主要成员对于中国耐药结核病基因型和表型的贡献并初探可能的潜在机制,为控制结核病在中国农村地区的传播提供线索。主要研究方法和结果如下:1.耐药结核病发生率和耐药结核病分布的横断面研究。通过对农村人群耐药结核病的现况调查,了解江苏和浙江两个社会经济发展水平不同样本县的结核病总耐药率,初/复治病人耐药率,MDR-TB率和各耐药专率。研究对象为浙江省德清县(2004年4月1日到2005年3月31日)和江苏省灌云县(2004年6月1日至2005年5月31日)县结核病防治所一年内痰培养阳性的肺结核病人。两县结核病防治所共登记结核病人784人,其中痰培养阳性结核病人399人(德清县182人,灌云县217人),包括283例新发结核病病人和116例复治结核病病人。经对分离获得的结核菌株进行抗结核一线药物的敏感试验检测发现,药物全敏感、MDR-TB和耐其他抗结核药物的病人数分别为148、58和193人。德清县和灌云县初治结核病人耐药情况分别为:总耐药率:50.4%(67人)和63.3%(95人);MDR-TB率:3.8%(5人)和14.7%(22人)。复治病人耐药情况分别为:总耐药率:67.3%(33人)和83.6%(56人);MDR-TB率:16.3%(8人)和34.3%(23人)。进一步分析生物学、社会人口学和行为等因素与耐药结核病的关系发现:病人的既往治疗史(≥4周/＜4周:德清县:OR=4.980;95%CI:1.509-8.031;灌云县:OR=3.941;95%CI:2.043-12.48)、病例接触史(是/否:德清县:OR=7.841;95%CI:1.712-25.30;灌云县:OR=4.801;95%CI:1.321-7.243)以及个人收入(高/低收入:德清县:OR=0.794;95%CI:0.038-0.927;灌云县:OR=0.150;95%CI:0.043-0.891)与MDR-TB人群分布有关。在灌云县,病人年龄(30～岁/60岁组:OR=2.748;95%CI:1.047-6.974),痰涂片结果(阳性/阴性:OR:5.721;95%CI:2.107-17.17)也与MDR-TB发生有关,男性是发生耐多药结核病的主要人群(男性/女性:OR=2.084,95%CI:1.061-6.349)。2.中国耐药结核菌基因型及其与耐药表型的相互关系的研究。本研究以351份可获得DNA的M.TB分离株及其宿主为研究对象,对与一线药物耐药有关基因rpoB、katG、inhA、rpsL和embB进行测序研究。研究发现,131株异烟阱(INH)耐药株中有81株(61.8%)在katG315位存在基因突变,主要的耐药相关基因型为315ACC(Thr)。利福平(RIF)耐药的分离株中,92.3%由rpoB基因核心区的突变所致,516、526和531密码子位的突变分别占RIF耐药菌株的12.3%、29.2%和56.9%;81.0%rpoB531Ser分离株为MDR-TB。115株链霉素(SM)耐药分离株中,60株出现了rpsL43AGG突变。另外42株乙胺丁醇(EMB)耐药分离株中有20株包含了embB306位的突变,85%embB306突变发生在多个药物耐药结核菌中。3.结核分枝杆菌(M.TB)尤其是耐药M.TB基因型的分布和成簇特征分析。采用MIRU、Spoligotyping和RFLP-IS6110作为基因分型手段,对已获得结核分枝杆菌DNA的351位病人及其M.TB分离株进行研究。首先采用Spoligotyping和MIRU进行基因分型和成簇性判别,然后对所有MIRU分型判断为成簇的分离株进行RFLP-IS6110二次分型。Spligotyping识别了15个簇和35株“唯一”分离株。在351株分离株中,243株分离株(69.2%)属于M.TB北京家族。MIRU基因分型识别了220种基因型,包括183种“唯一”基因型以及37个簇(包括168株分离株)。最大的一组MIRU簇包含38(10.8%)株分离株,拥有相同的MIRU基因型223325173533(山东簇)。RFLP进一步在MIRU簇分离株中定义了31个簇,尤其将最大MIRU簇细分成9个亚组,其中最大亚组包括4个分离株。4.耐药M.TB成簇性影响因素分析。本部分研究以MIRU和IS6110-RFLP联合基因分型定义的62株耐药“成簇”M.TB分离株和161株“唯一性”耐药M.TB分离株及其宿主为研究对象,比较成簇组和非成簇组耐药病人的人口学、社会经济水平特征及其就医行为。结果发现,灌云县病人比德清县病人更容易发生耐药菌株成簇现象(34.1%.vs.18.2%;OR=3.311:95%CI:1.498-7.319)。30-60岁年龄组相比60岁以上人群所感染的M.TB更容易成簇(30～岁/60～岁年龄组:34.7%.vs.19.0%;OR=2.326;95%CI:1.039-5.393)。另外痰涂片阳性的耐药M.TB比涂阴耐药M.TB更易出现成簇(涂阳/涂阴:32.2%.vs.18.9%:OR=2.242;95%CI:1.010-4.977)。北京家族中山东簇分离株更加容易成簇(山东簇/非北京家族:67.7%.vs.15.3%;OR=6.681;95%CI:3.258-15.02)。5.中国农村地区耐药结核病传播模式研究。本部分研究以MIRU和RFLP联合基因分型定义的成簇病人为研究对象,通过案例追踪,探讨农村耐药结核病传播的可能途径。研究发现,中国农村地区耐药结核病传播呈现以村落为单位的小范围、散发特点。30-60岁人群和复发病人可能是结核病尤其是耐药结核病的主要传染源。同时,偶然接触导致的疾病传播在结核菌传播中占有较大比重。6.北京家族及其成员高耐药基因表型和基因型特征的潜在原因初探。通过比较243株Spoligotyping定义的北京株M.TB和108株非北京株耐药基因型和表型,研究北京家族分离株尤其是其中流行优势成员山东簇与耐药基因表型和基因型的关系,并尝试从北京家族修复基因SNP多态性以及IS6110的插入片段数分析北京家族及其成员高耐药基因表型和基因型特征的潜在原因。研究发现,北京株及其成员山东簇与MDR-TB(北京家族/非北京家族:18.5%.vs.7.4%:OR=2.723,95%CI:1.351-6.733;山东簇/其他北京家族:44.7%.vs.13.7%;OR=6.18;95%CI:2.68-14.23)和相应的rpoB和katG联合基因突变(北京家族/非北京家族:13.6%.vs.5.6%;OR=2.553,95%CI:1.031-6.324;山东簇/其他北京家族:34.2%.vs.9.8%;OR=5.901;95%CI:2.581-13.46)之间存在明显的联系。修复基因多态性变化以及IS6110插入数量可能与北京株以及山东簇发生耐药基因型变化、获得耐药表型有关。更多还原

【Abstract】 Since 1990s,DOTS based strategy has been expanded to most of areas in China, providing the free anti-YB therapy to more than 1,300 million infectious pulmonary tuberculosis patients and subsequently helping China reaching the 70%of cure rate set by WHO in 2006.But considering the disease burden,China remains to be one of countries with the highest TB burden,only second to India.China has owned 16%of incident TB cases（1.58/9.88million） worldwide,with smear positive TB rate and active TB rate being respectively 46/100,000 and 101/100,000 in 2004.The control of tuberculosis in China has been further complicated and threatened by an increasing incidence of drug-resistant tuberculosis especially including mulit-drug-resistant tuberculosis（MDR-TB） in last ten years.MDR-TB is the pattem of drug resistance to at least isoniazid and rifampicin.A recent global survey of MDR-TB incidence estimated that an average of 2.7%of all new cases with no TB history,18.5%of previously treated and 4.3%of all cases had MDR-TB in 2004[2].Indeed,the MDR-TB incidence estimates for China in 2004 was considerably higher than the global average with 5.3%among cases with no history of TB,27.2%among previous treated cases.The 4^th TB epidemiological study reported that 18.7%patients were resistant to at least one of the first-line anti-TB drugs and 6% of TB patients had MDR-TB.The monitoring data from different sources has demonstrated the hotspot of drug resistant TB in some Chinese areas.Drug resistant TB could directly cause the high prevalence and mortality of TB and meanwhile the elongation of treatment period and increase in treatment cost.Setting in two DOTS covered rural counties respectively from Jiangsu and Zhejiang provinces,this study attempted to describe the prevalence and transmission of drug resistant TB in rural area of Eastem China from multi-displinary perspective in terms of field epidemiology and molecular epidemiology:to describe the prevalence of drug resistant TB and socioeconomic factors influencing its occurrence by the cross sectional study;to investigate the genetic diversity of prevailing M.TB strain and explore the transmission mechanism of drug resistant TB by combining all the available genotyping methods including MIRU, Spoligotying and IS6110;to identify the drug resistance genotype and its association with phenotype by direct DNA sequencing;to determine the transmission pattern of drug resistant TB strain in rural China through tracing the clustered cases:to study the contribution of Beijing genotype and its predominant member to the genotype and phenotype of drug resistant TB in rural China and uncover the mechanism behind them.1.A cross-sectional study was conducted in two rural counties in Eastern China: Deqing with over 10 years’ DOTS implementation and Guanyun under its second year of DOTS.The subjects were all culture-positive pulmonary TB patients newly diagnosed or retreated during 12 months of 2004 to 2005.The proportion method was used for drug susceptibility testing.Among the 399 subjects,283 were new TB cases and 116 were previously treated.The rates of overall resistance in new cases were 50.4%（67） and 63.3%（95） respectively in Deqing and Guanyun,and 67.3%（33） and 83.6%（56） respectively in previously treated cases.The rates of MDR-TB in new cases were 3.8%（5） in Deqing and 14.7%（22） in Guanyun（p=0.0018）,and 16.3% （8） and 34.3%（23） in previously treated cases（p=0.0305）.Furthermore,the comparision of socio-demographic and medical characteristics was made between 58 MDR-TB patients,193 other drug resistant and 148 pan-drug sensitive TB patients.Compared to pan-drug sensitive patients,both in two counties, previous treatment history（≥4weeks/＜4weeks:Deqing:OR:4.980;95%CI:1.509-8.031;Guanyun:OR:3.941;95%CI:2.043-12.48）,case contact（yes/no:Deqing: OR:7.841;95%CI:1.712-25.30;Guanyun:OR:4.801;95%CI:1.321-7.243） and individual income（high/low income:Deqing:OR:0.794;95%CI:0.038-0.927; Guanyun:OR:0.150;95%CI:0.043-0.891） were associated with MDRTB.Only in Guanyun with shorter duration NTP-DOTs,were age（30～year/60～year group:OR: 2.748;95%CI:1.047-6.974） and smear positive（smear positive/negative:OR:5.721; 95%CI:2.107-17.17） related to MDRTB.There was higher proportion of MDRTB among male patients（male/female:OR:2.084,95%CI:1.061-6.349）.It was concluded that Socioeconomic（age,sex and individual income） and clinic（case contact and previous treatment history） characteristics were risk factors related to presence of drug resistance TB.The vulnerable population（the poor and the elder） should become the concern of the TB control.2.DNA sequencing on the hotspot of resistance related genes was applied to investigate the drug-resistance genotype and its association with the corresponding drug resistance phenotype.81（61.8%） of 131 INH resistant isolates had single-nucleotide substitution in codon 315 of katG with 315ACC（Thr） responsible for 49.6%of INH-resistance.Drug resistance to RIF was mostly due to the genetic mutation of rpoB gene,where the single-nucleotide substitution in codon 516,526 and 531 took account respectively for 12.3%,29.2%and 56.9%.81.0%isolates with rpoB531Set were MDR-TB.Meanwhile,the DNA sequencing analysis of rpsL43, revealed resistance mutations in 60 of 115 SM resistant isolates totally due to a single base change（43rpsLAGG）.While spot mutation related to mono-drug SM resistance represented a largest proportion（51.3%）.Among 20 of 42 EMB-resistant isolate containing the genetic mutation in embB306,most of embB306 mutations were observed in the EMB-resistant M.TB isolate simultaneously resistant to other first-line anti-TB drugs.3.Three genotyping methods,including mycobacterial interspersed repetitive unit（MIRU） typing,Spoligotyping,and IS6110 restriction fragment length polymorphism（RFLP） typing,were used in this study.All 351 MTB isolates were first molecularly characterized by Spoligotyping and MIRU.Each MIRU cluster was further genotyping by IS6110 RFLP（i.e.MIRU plus IS6110） for clustering analysis. Spoligotyping identified 243 isolates（69.2%） that presented the Beijing family Spoligotype.MIRU typing determined 220 genotypes from all isolates in this study, including 183 unique patterns and 37 clusters（including 168 isolates）.The largest MIRU cluster contained 38（10.8%） isolates,sharing an identical MIRU genotype 223325173533,which has been referred to as the "Shandong cluster" in a previous study.Again,all isolates from the "Shandong cluster" were members of the Beijing family.Shandong cluster was further distinguished into 9 sub-clusters by IS6110 RFLP.The largest MIRU/IS6110 cluster had 4 isolates.4.Based on the MIRU-IS6110 pattern,the 62 isolates was defined as the clustered and 161 as the unique.The comparison of host’s socio-demographic and medical characteristics between these two groups revealed the drug resistant isolates in Guanyun were more likely to be clustered compared to Deqing（34.1%.vs.18.2%; OR:3.311;95%CI:1.498-7.319）.In addition,the population aged 30-60 year was in high risk of recent transmission causing the disease（30～year/60～year age group: 34.7%.vs.19.0%;OR:2.326;95%CI:1.039-5.393）.The smear positive was the factor independent of disease due to the recent transmission(（Postive/Negative.32.2%.vs. 18.9%;OR:2.242;95%CI:1.010-4.977）.The Shandong cluster was more likely to develop the disease through the recent transmission（67.7%.vs.15.3%;OR:6.681; 95%CI:3.258-15.02）.5.Those with the clustered isolated was investigated to describe the transmission pattern of drug resistant TB through case tracing.The transmission pattern of drug resistant TB was presented by the sporadic distribution in small group.The population aged 30-60 years and the previously treated patients were the main sources of infection.Meanwhile,the causal contact contributed a lot to the recent transmission of TB.6.Targeting the 243 Beijing isolates available,this part of study was to investigate the contribution of Beijing family and its predominant member to drug resistance genotype and phenotype.The attempt was made to explore the mechanism behind the connection between Beijing family and drug resistance in terms of the polymorphism of repair gene and bands of IS6110 insertion.The Binary Logistic Regression Model adjusted by age,sex,county of the subjects was applied to analyze the association between drug resistance,gene mutation and genotypes.Beijing family isolates presented increased risks to be MDR-TB（OR:2.723,95%CI:1.351-6.733） and have katG and rpoB mutations（OR:2.553,95%CI:1.031-6.324） compared with non-Beijing family isolates.Clustering analysis by MIRU plus IS6110 RFLP revealed that Beijing family isolates were more likely to be clustered.The repair gene and bands of IS6110 insertions proven to play a role in the acquiring the genotype and phenotype of drug resistance.更多还原