【作者】 林辉；

【导师】 曹佳；

【作者基本信息】 第三军医大学 ， 卫生毒理学， 2008， 博士

【摘要】 耐药结核病尤其耐多药结核病(Multi-drug resistance tuberculosis,MDR-TB)的发生和流行是自20世纪80年代中后期以来结核病疫情回升的主要原因之一。预防和控制耐(多)药结核病已经成为当前结核病控制的主要任务,而了解结核病耐药疫情基线数据和流行病学特征是制定预防和控制耐药结核病的重要依据。世界卫生组织和国际防痨和肺病联合会(WH0/IUATLD)为了解全球耐药结核病总的分布情况,于1994年启动全球结核病耐药性监测计划,在1994~2002年间,对全球114个国家和地区进行了抗结核一线药物(链霉素、异烟肼、利福平、乙胺丁醇)的耐药性的调查,覆盖全球约50%的人口,其调查结果显示全球耐(多)药结核病呈不断蔓延扩散的趋势,发展中国家和地区上升尤为显著。我国属于耐药程度较高的国家之一,被WHO列为“特别引起警示的国家和地区”和MDR-TB的“热点”地区之一。流行病学调查显示:通常经济和卫生、医疗条件等较差的地区,耐药结核病疫情尤为严重,同时结核病疫情和耐药结核病的流行病学特征存在明显的地区差异。重庆地处西南,农村人口多,经济不够发达,近10年尚无大样本量的耐(多)药结核病相关流行病学研究调查报道,因此了解本市耐(多)药结核病的相关基线资料和流行病学特征对于促进耐(多)药结核病的控制有重要意义。本研究第一部分,即是对在2003年~2006年在重庆市法定结核病防治机构(结核病防治所和胸科医院)连续收集的痰培养阳性的1089例结核病病例的耐药情况和流行病学特征进行分析。异烟肼(Isoniazide,INH)和乙胺丁醇(ethambutol,EMB)是WHO推荐使用的一线抗结核组合药物之一。近年WHO/IUATLD在全球的结核病耐药监测结果显示:全球INH平均耐药率为5.6%,大多数国家和地区INH耐药率呈现逐年上升趋势,且在四种一线抗结核药物(INH,RFP,SM和EMB)中,INH耐药率在各国和地区均排列第一或第二位,我国INH耐药率为17.6%,耐药水平较高。全球平均EMB耐药率为0.8%(0~24.8),尽管总体耐药水平较低,但呈逐年快速上升趋势,我国EMB耐药率(1.5%)显著高于全球平均EMB耐药水平。INH和EMB耐药率的高企和升高,将明显影响WHO推荐的以此为主要组合药物方案治疗结核病的疗效。从降低耐药结核病病人死亡、减少传染源、缩短传染期的角度来看,早期诊断、早期选择敏感药物治疗耐药结核病病例是预防和控制耐(多)药结核病和减少卫生资源浪费的重要措施之一。但目前以培养为基础的药敏检测方法需要较长的时间(1~3月)才能得到结果,难以实现早期诊断和及时指导临床用药的目的。随着分子生物学理论和技术的发展,结核分枝杆菌INH耐药和EMB耐药的分子机制得到一定程度的阐明,目前研究已经证实结核分枝杆菌内编码活化INH所必需的过氧化氢酶-过氧化物的katG基因(尤其katG315)的变异是INH耐药性产生的最主要分子机制,但katG315在不同地区的INH耐药结核分枝杆菌中的变异率存在明显差异,从26%至95%不等。结核分枝杆菌中编码阿拉伯糖基转移酶的embB基因突变(尤其306位密码子)是引起结核分枝杆菌乙胺丁醇耐药的最主要原因, embB306突变覆盖约50%~70%的EMB耐药菌株,是目前与EMB耐药最相关的分子生物学标记物。部分地区已将katG315和embB306突变作为耐药分子标记物,采用PCR-FRLP和PCR-SSCP等方法进行临床INH和EMB耐药菌株的检测,实现了INH和EMB耐药病例的早期诊断和及时指导临床治疗,具有较高的特异性和敏感性,研究证明katG315、embB306分别在INH、EMB耐药结核杆菌中突变率较高的地区,这些快速、价廉的分子生物学方法在临床上应用是可行的。由于分子生物学检测方法是通过基因型来推测细菌耐药的表型,且katG基因和embB基因变异具有明显的地域分布特点,重庆地区尚无这两个基因在结核分枝杆菌中的突变谱、突变率的研究报道,katG315和embB306是否是本地区INH耐药、EMB耐药结核分枝杆菌的主要分子机制?是否可分别用于筛选或检测INH耐药、EMB耐药结核病的分子标记物?有待研究阐明。本研究第二部分和第三部分采用测序方法分别对重庆不同耐药组合的97株结核分枝杆菌中的katG基因和101株结核分枝杆菌中的embB基因的多态性及它们与耐药相关性进行了分析,将有助于阐明以上问题。主要结果和结论如下:一、第一部分重庆市2003 ~2006年结核病耐药情况的流行病学调查1.重庆市结核病总耐药率和耐多药率分别是27.6%(301/1089)和7.3%(79/1089);其中初治耐药率和耐多药率分别是22.7%、4.1%,复治耐药率和耐多药率分别是56.2%、26.5%。整体耐药水平与国内外监测结果比较,处于中等水平。2. SM、INH、RFP、EMB耐药率分别是16.3%、14.0%、10.7%和4.7%;近3年RFP、EMB耐药率和耐多药率呈逐年上升趋势。3.耐(多)药病例分布无明显性别和年龄特征,但与用药史、经济条件差(居住乡村、可支配年收入低于平均水平)存在较强联系。提示耐(多)药结核病防治应重点加强直接面视下督导短程化疗( Directly Observed Treatment Short course, DOTS)方案的实施和经济条件较差的结核病病例的规律全程治疗。4.耐药病例的分布特征提示:重庆地区可能存在一定范围、一定程度的耐药结核分枝杆菌的传播,应加强结核病的耐药监测尤其分子水平的监测,有利于早期发现和预防耐药结核分枝杆菌大规模的扩散,对于控制耐(多)药结核病疫情有重要意义。二、第二部分重庆市97株结核分枝杆菌的katG315突变特征及其与异烟肼耐药相关性的研究1.重庆地区INH耐药结核分枝杆菌中katG315突变谱包括S315T、S315N和S315三种类型,总突变率为75.5%(37/49),其中81.1%(30/37)为S315T突变型,INH敏感菌株中未见katG315突变。katG315突变是本地区结核分枝杆菌INH耐药的主要分子机制。2.以katG315突变做为诊断INH耐药菌株的标记物,与传统药敏试验比较,灵敏度为75.5%,特异度为100%,准确度为87.6%(85/97),其他各项评价指标均提示:在本地区,该指标用于INH耐药菌株的快速诊断具有较好的的临床应用价值。3. katG315在高度耐药(1ug/ml)和耐多药菌株中的突变率分布分别显著高于低度耐药(0.2 ug/ml)和单耐INH菌株,且与耐药数量有关,一定程度上表明katG315突变与耐药程度有关。4. katG突变率分析显示:katG315变异是本地区INH耐药产生的主要分子机制,24.5%的INH耐药株未见katG315变异,表明在本地区还存在其他INH耐药的分子机制,对于本地区结核病、耐药结核病的防治有一定参考价值。三、第三部分重庆101株结核分枝杆菌的embB306突变特征及其与乙胺丁醇耐药相关性的研究1. embB306在EMB耐药菌株中突变率有明显地域差异,重庆地区EMB耐药菌株中embB306突变率66.7%,34/51显著高于EMB敏感菌株的突变率(6.0%,3/50)(P〈0.01),与传统药敏实验比较,embB306突变作为EMB耐药标记物检测EMB耐药的灵敏度为66.7%(34/51),特异度为90%(45/50),准确度为78.2%(79/101)。提示在没有更好标记物情况下,embB306突变应用于本地区的EMB耐药菌株的检测有一定临床意义。2. embB306在EMB耐药MDR菌株中突变率为84.0%(21/25)显著高于非MDR菌株中的突变率(50.0%,13/26),且突变率随耐药数量增加而增加,提示embB306突变与耐药程度有关。3. embB突变谱较广泛,embB306突变覆盖66.7%(34/51)的EMB耐药菌株,提示embB306突变是本地区EMB耐药产生的主要分子机制,其他分子机制可能与大量稀少突变及其联合作用有关。总之,通过本研究获得了重庆结核病耐药基线资料和耐药结核病的主要流行病学特征;同时明确了KatG315和embB306突变分别是本地区结核分枝杆菌INH、EMB耐药的主要分子机制,它们作为INH、EMB耐药快速检测的分子标记物具有一定临床应用价值。该研究结果对于本地区结核病、耐药结核病的防治有一定参考价值。更多还原

【Abstract】 The incidence and infection of drug resistance tuberculosis (DR-TB), especially for the multi-drug resistance tuberculosis (MDR-TB) is one of the major cause to revive the epidemic of tuberculosis. WHO and IUATLD launched a global program for surveillance of drug resistance in 1994 to identify global DR-TB distribution, and conducted a resistance investigation on the first line drugs, including streptomycin(SM), isoniazid(INH) rifampicin (RFP) and ethambutol(EMB), among114 countries and area in the world from 1994 to 2003 to disclose the growing tendency of MDR-TB in developing countries. China is on the list of“Countries and Districts need more attention”proposed by WHO due to the severe situation of drug resistance. MDR-TB problem in China is also a“hot topic”.The main task for TB control at present is to prevent and control MDT-TB, for which, the baseline information and epidemic characteristics of DT-TB shall be collected for reference to make preventive or administrative policies. However, both DR-TB information and epidemic characteristics show regional differences. Located in southwest, Chongqing is confronted with the pressure of large rural population and relatively underdeveloped economy. In the last decade, there is no local report on (M)DR-TB based on large sample size. In a word, the baseline information and epidemic characteristics of local (M)DR-TB is critical to DR-TB control. Section 1 in the present study disclosed smear positive samples sequentially collected by authorized local TB prevention organization (Tuberculosis Prevention and Control Institute, chest hospital etc.), total 1089 cases, and the analysis of drug resistant information and epidemic characteristics thereof.INH and EMB are the first line TB resistance drugs recommended by WHO and one of the indispensable compound drugs for TB treatment. According to the global surveillance report on drug resistance tuberculosis by WHO, the resistance rate of INH is rising yearly around the world. Of all the four TB drugs (INH, RFP, SM and EMB), the resistance rate of INH is the highest or the second highest, 5.6% on average. China’s INH resistance rate is 17.6%, rather a high level. Generally speaking, the global EMB resistance rate is low, only 0.8% (0~24.8) on average, but rising rapidly every year. China’s EMB resistance rate (1.5%) is higher than the global average value.To reduce death rate of DR-TB patients, infection sources and infection period, diagnosis and selection of sensitive drugs at the early stage is the most important method to help TB patients. However, the present drug sensitivity testing method based on culture is too time-consuming (1-3months) to be used in clinic timely. The developing molecular biological theories and techniques shed some light on the molecular mechanism of INH resistance and EMB resistance of mycobacterium tuberculosis. Up to now, researchers have proved that the mutation katG genes (especially katG315), an indispensible catalase- peroxide to INH activation by internal-coding of mycobacterium tuberculosis, is the major cause for INH resistance. Also the mutation rates of katG315 in INH-resistant mycobacterium tuberculosis shows regional differences (26%~95%). The mutation of embB (especially the 306th codon), the gene that is responsible for coding arabinose glycosyltransferase is the major cause to lead EMB resistance. embB 306 mutation was detected on 50%~70% EMB-resistant stains, so it is regarded as the molecular biological marker closely related to EMB resistance. katG315 mutation and embB306 mutation are used as the clinic testing methods for INH and EMB resistance respectively in some regions, by which the diagnosis and therapy of INF or EMB resistance are realized as well as showing high sensitivity and specificity. So it is safe to declare the feasibility of applying those rapid and economical molecular biological methods to clinic.Molecular biology is to estimate the phenotype of drug resistance through genotype, and the gene mutation of katG and embB shows significant regional differences. By now, there is no report on the mutation rate or mutation spectrum of those two genes in mycobacterium tuberculosis, whether katG315 and embB306 can be used as the marker to screen and test INH and EMB resistance of mycobacterium tuberculosis needs further exploration. Section 2 and Section 3 in this study used sequencing method to analyze the polymorphism of katG in 97 strains and embB in 101 strains of local patients by different compound drugs, and set a base to form a rapid molecular biological method to test INH resistance or EMB resistance in Chongqing. The main results and conclusions are as follows:1. The general DR-TB rate and MDR-TB rate is 27.6% (301/1089) and 7.3% (79/1089); the DR-TB rate and MDR-TB rate for new case is 22.7% and 4.1%; and the DR-TB rate and MDR-TB rate for re-treatment is 56.2% and 26.5%. In general, Chongqing’s DR-TB level is in the middle level compared to the domestic or foreign surveillance results.2. The resistance rates for SM, INH, RFP and EMB are 16.3%, 14.0%, 10.7% and 4.7% respectively. In the recent three years, RFP resistance, EMB resistance and multi-drug resistance are constantly rising.3. Multi-drug resistance cases show no obvious gender of age difference, but they are closely related to chemotherapy history and living conditions (rural environment, annual income lower than average level etc.)4. The distribution of (M)DR-TB cases indicated that epidemic spreading of (M)DR-TB might exist within a certain scope to some extent in some area of Chongqing, so it is necessary to reinforce the surveillance of drug resistance, especially at the molecular level, in order to diagnose the expansion of mycobacterium tuberculosis at an early stage, which is also important to control the epidemic situation of (M)DR-TB.5. katG315 mutation spectra from INH resistant strains in Chongqing can be divided into three types: S315T, S315N and S315, with the general mutation 75.5% (37/49), including 81.1% (30/37) S315 mutation. katG315 mutation was not detected in INH sensitive strains.6. Compared to the traditional drug sensitivity test, , the method by using katG315 as the INH-resistant marker showed a high sensitivity 75.5%, specificity 100% and accuracy 87.6% (85/97). And according to other indicators, such method showed a fair clinic application value.7. katG315 mutation in MDR-TB strains was significantly higher than that in INH-resistant strains, and related to drug resistance quantity, so it is suitable to be used as the molecular marker for MDR-TB test.8. The analysis of katG mutation spectrum indicated that katG315 mutation is the major molecular mechanism to generate the local INH resistance. No katG315 mutation was detected in 24.5% INH resistant strains, indicating that there were some other mechanisms in the local area to generate INH resistance. The influence of mutation other rare point positions on the generating process of INH resistance needs further study.9. embB306 mutation rate in EMB-resistant strains (66.7%) was significantly higher than that in EMB-sensitive strains (60.0%). Compared to the traditional drug sensitivity test, the method by using embB306 as the EMB-resistant marker showed a high sensitivity 66.7%, specificity 90% and accuracy 78.2%, indicating that the rapid method by using embB306 mutation to test EMB resistant strains is applicable to the clinic.10. embB306 mutation rate in EMB-resistant MDR strains (84.0%) was significantly higher than that in non-MDR strains (60.0%), and ascending with the growth of drug resistance quantity, indicating that embB306 is more suitable for testing EMB-resistant MDR strains as a marker.11. embB mutation spectrum is in broad distribution. In the present study, embB306 mutation covered 66.7% EMB-resistant strains, indicating that embB306 mutation is the major molecular mechanism to generate EMB resistance in Chongqing, and that other molecular mechanisms are probably related to a number of rare mutations and the common results of all those rare mutations.In conclusion, the present study outlined the baseline information and the major epidemic characteristics of (M)DR-TB in Chongqing, and identified that katG315 and embB306 are the major molecular mechanisms for INH resistance and EMB resistance respectively, so the method by using INH or EMB as the marker for rapid test of drug resistance shows a fair clinic application value. Besides, the results of this study also provides reference for prevention and control of TB, and (M)DR-TB in Chongqing更多还原